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Demonstration of amelogenin in the enamel-free cusps of rat molar tooth germsImmunofluorescent and immunoelectron microscopic studies.

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THE ANATOMICAL RECORD 233:588-596 (1992)
Demonstration of Amelogenin in the Enamel-Free Cusps of Rat
Molar Tooth Germs: lmmunof luorescent and lmmunoelectron
Microscopic Studies
TETSUICHIRO INAI, KENGO NAGATA, TOSHIO KUKITA, AND KOJIRO KURISU
Second Department of Conservative Dentistry (T.I.) and Second Department of Anatomy
(K.N., T.K., K.K.), Faculty of Dentistry, Kyushu University,Fukuoka, Japan
ABSTRACT
The enamel-free cusps of 1-4-day-old r a t mandibular first molars
were investigated using the monoclonal antibody En3 against rat amelogenin a t
light and electron microscopic levels in order to clarify whether the enamel-free
cusp is virtually devoid of enamel.
At 1 day after birth, there were presecretory ameloblast-like cells (PALCs),
which were short and were not polarized, a t the cusp tips. They were close to the
outer enamel epithelium. Hematoxylin positive enamel matrix was not distinctly
observed in the enamel-free cusp by light microscopy, but almost continuous immunofluorescence for amelogenin was detected a t the interface between PALCs
and dentin. The penetration of immunopositive material toward the dental pulp
was also observed in the enamel-free cusp.
At 4 day after birth, both in the frontal section and in the horizontal section,
almost continuous immunof luorescence was recognized a t the interface between
PALCs and dentin in the enamel-free cusp. The penetration of amelogenin toward
the dental pulp was not seen in the enamel-free cusp. By immunoelectron microscopy, immunolabelling was recognized in the Golgi apparatus of PALCs, in a layer
of amorphous material a t the interface between PALCs and dentin, and in stippled
material-like substance in the intercellular space between PALCs. Although no
basement membrane was observed beneath PALCs, they did not have Tomes’
processes.
These investigations suggest that PALCs in the enamel-free cusp differentiate
into the secretory cells and that they can synthesize and secrete the amorphous
material containing amelogenin a t the interface between PALCs and dentin. The
penetration of amelogenin toward the dental pulp might play a role in the interaction between PALCs and odontoblasts in the enamel-free cusp and/or the initiation of mineralization of predentin. o 1992 Wiley-Liss, ~ n c .
Tooth crown formation results from reciprocal and
interdependent epithelial-mesenchymal interactions
(Kollar, 1972, 1983; Ruch and Karcher-Dijuricic, 1975;
Slavkin et al., 1984; Ruch, 1985). The cells of the inner
enamel epithelium of the enamel organ differentiate
into secretory ameloblasts that produce enamel,
whereas ectomesenchymal cells adjacent to the epithelium differentiate into odontoblasts that produce predentin and dentin. However, there are non-enamel
bearing regions at the occlusal cusps of molar teeth of
the rat (Addison and Appleton, 1921; Mellanby, 1939;
Lefkowiz et al., 1953) and of the mouse (Gaunt, 1956;
Cohn, 1957). It has been reported that although mesenchymal cells differentiate into odontoblasts and consequently normal dentinogenesis occurs in the enamelfree cusp, the cells of the inner enamel epithelium in
the enamel-free cusp fail to differentiate into secretory
ameloblasts and to secrete enamel matrix. However,
some workers have reported that the cells of the inner
enamel epithelium in the enamel-free cusp differentiate into secretory cells which can secrete enamel ma0 1992 WILEY-LISS. INC.
trix (Johannessen, 1961; Sutcliffe and Owens, 1980,
1981; Diab and Zaki, 1985; Nakamura et al., 1987;
Sakakura et al., 1989). Nakamura et al. (1986) observed discrete immunoreactivity for polyclonal antibodies against mouse amelogenin at the interface between the cells of the inner enamel epithelium and
adjacent dentin in the enamel-free cusps of mouse molars using indirect immunofluorescent microscopy.
However, a precise immunohistochemical study in the
enamel-free cusp using monoclonal antibodies against
distinct enamel proteins at light and electron microscopic levels has not yet been reported.
In the present study, we examined the enamel-free
cusps of rat molar tooth germs using the monoclonal
Received April 16, 1991; accepted January 8, 1992.
Kojiro Kurisu’s present address is First Department of Oral Anatomy, Faculty of Dentistry, Osaka University, 1-8,Yamadaoka, Suita,
Osaka 565, Japan. Address reprint requests there.
Figs. 1-3 (legend on following page).
590
T. INAI ET AL
antibody En3 (Inai et al., 1991) directed against rat
amelogenin a t light and electron microscopic levels.
MATERIALS AND METHODS
We investigated the enamel-free cusps of rat mandibular first molars. The immunohistochemical localization of amelogenin in the enamel-free cusp was examined a t light and electron microscopic levels using
the monoclonal antibody En3 (Inai et al., 1991) which
recognizes rat amelogenin.
IrnrnunofluorescenceProcedure
The 1-4-day-old rats after birth were perfused with
periodate-lysine-paraformaldehyde fixative (McLean
and Nakane, 1974) through the left ventricle and the
dissected mandibles were immersed in the same fixative for about 12 hours at 4°C. After decalcification by
10% ethylenediamine tetraacetic acid (EDTA) for 1
week at 4"C, the specimens were embedded in paraffin.
Sections mounted on glass slides were incubated with
the monoclonal antibody En3 for 1 hour a t room temperature. Control sections were treated with either
normal mouse serum (Zymed Lab., Inc., South San
Francisco, CA) or PBA (phosphate-buffered saline
(PBS) containing 1%chick albumin) instead of En3.
After repeated rinsing with PBS, the tissue sections
were incubated with fluorescein isothiocyanate (FITC)
labelled goat anti-mouse p-chain antibody (Zymed
Lab., Inc.) for 30 minutes at room temperature. The
entire procedure was carried out in a moist chamber to
prevent the samples from drying. Finally the sections
were washed extensively with PBS and sealed with
PBS-buffered glycerol to which p-phenylenediamine
had been added to decrease fluorescent fading as described by Johnson and Araujo (1981). The specimens
were then examined with a Zeiss fluorescence microscope. After recording of the immunofluorescent reac-
Figs. 1-4. Frontal and horizontal sections of mandibular first molars of 1-4-day-old rats. After recording of the immunofluorescent
reaction using the monoclonal antibody En3, the same sections were
stained with hematoxylin and eosin (H + E) for histological observations.
Fig. 1. A micrograph of the frontal section of 1-day-old rat mandibular first molar stained with H + E. Ameloblasts in the cusp are close
to the outer enamel epithelium (arrows). x 75.
Fig. 2. Higher magnification of a n area in a square shown in Figure
1, stained with En3 (a) and H + E (b). The nuclei of most of ameloblasts in the cusp are migrating to the proximal cytoplasm and some
of them are secreting hernatoxylin positive enamel matrix (arrowhead
in b). Some ameloblasts (arrow in b) at the cusp tip which are close to
the outer enamel epithelium have not been polarized. Almost continuous immunofluorescence for En3 is observed at the interface between ameloblasts and dentin. Note the staining for En3 in dentin,
predentin, and odontoblast layer (arrows in a). A ameloblasts; 0:
odontoblasts. X 270.
Fig. 3. Micrographs of the frontal section of 4-day-old rat mandibular first molar stained with En3 (a) and H + E (b). No distinct
enamel matrix is observed a t the interface between presecretory
ameloblast-like cells (PALCs),which are short and not polarized, and
dentin (D), but there are a few discrete thin layers stained with hernatoxylin between PALCs and dentin (arrows in b). Almost continuous immunofluorescence is recognized at the interface between
PALCs and dentin, and it continues to enamel matrix (E) with imrnunoreactivity. x 320.
tion using En3, the same sections were stained with
hematoxylin and eosin (H + E) for histological observations.
lrnrnunoelectron Microscopy
The 4-day-old rats were perfused through the left
ventricle with a mixture of 1%glutaraldehyde and 3%
paraformaldehyde in 0.1 M cacodylate buffer containing 0.05% CaCl,, pH 7.4. The mandibles were dissected
and immersed in the same fixative for about 6 hours a t
4°C. After decalcification in 10% EDTA for 1 week at
4"C, the mandibular first molars were cut with a Microslicer (Dosaka EM Co., Kyoto, Japan) into thick sections of 200-300 pm which were postfixed in osmium
tetroxide reduced with potassium ferrocyanide (Karnovsky, 1971) for 2 hours at room temperature in the
dark. The thick sections were washed in a sodium cacodylate buffer, dehydrated in a graded series of ethanol, and embedded in Epon 812. Ultrathin sections
were mounted on nickel grids and floated on drops of a
saturated aqueous solution of sodium metaperiodate
for 1 hour and on PBL (0.1 M PBS containing 1%bovine serum albumin [BSAI and 0.1 M lysine-HC1) for
30 minutes. They were then treated with En3 antibody
for 1 hour at room temperature. Control sections were
treated with either normal mouse serum or PBL instead of En3. After repeated rinsing with PBS, the sections were incubated with colloidal gold labelled goat
anti-mouse p-chain antibody. Colloidal gold labelling
of goat anti-mouse p-chain antibody (Zymed Lab., Inc.)
was carried out as described by Slot and Geuze (1984).
The sections were then washed three times for 5 minutes each with PBS and finally again in distilled water.
Throughout the entire procedure, care was taken to
prevent the sections from drying by keeping them in a
moist chamber. The sections were finally stained with
uranyl acetate and lead citrate and then were examined with a JEOL 200CX electron microscope.
Fig. 4. Micrographs of the horizontal section of 4-day-old rat mandibular first molar stained with En3 (a) and H E (b).A thin layer of
hematoxylin positive enamel-like matrix (arrows in b), which continues to enamel matrix (E), is detected beneath presecretory ameloblast-like cells (PALCs), but there is a region where no enamel-like
matrix is detected below PALCs (arrowhead in b). Continuous immunofluorescence is observed clearly between PALCs and dentin in a.
x 250.
+
Fig. 5. Micrographs of control section, incubated with PBA instead
of En3 (a)and stained with H + E (b),of 1-day-old rat mandibular first
molar. Faint background staining is observed in predentin. A: ameloblasts; D: dentin; PD: predentin; 0: odontoblasts. x 270.
Figs. 6-1 0. Imrnunoelectron micrographs stained with the monoclonal antibody En3. Rat mandibular first molars of day 4 were prepared and immunostained with post-embedding method as described
in Materials and Methods.
Fig. 6. Control section of enamel matrix (EM) and Tomes' process
(TP) of secretory ameloblast in the area other than the enamel-free
cusp incubated with PBL in place of En3. No specific labelling is
observed. x 46,000.
Fig. 7. Almost the same area as shown in Figure 6 stained with En3.
Enamel matrix (EM) and secretory granule (arrow) in Tomes' process
ITP) are labelled. x 46,000.
Figs. 4-7.
592
T. INAI ET AL.
RESULTS
lmmunofluorescent Observations
Figure 1 shows the frontal section of 1-day-old rat
mandibular first molar. Ameloblasts at the cusp tip
were close to the outer enamel epithelium. Higher
magnification of the cusp of Figure 1 is shown in Figure 2. Most of ameloblasts in the cusp were columnar
and polarized, and some of them were secreting a thin
layer of hematoxylin positive enamel matrix (Fig. 2b).
However, ameloblasts a t the cusp tip, those that were
close to the outer enamel epithelium, were not polarized and the morphological features of them resembled
that of presecretory ameloblasts (Fig. 2b). In this article, we call them “presecretory ameloblast-like cells
(PALCs).”Odontoblasts in the cusp were well differentiated and produced predentin and dentin (Fig. 2b). Almost continuous immunofluorescence for the monoclonal antibody En3 was detected at the interface
between ameloblasts including PALCs and dentin (Fig.
2a). The penetration of immunopositive material from
ameloblasts including PALCs into the dental pulp was
also observed (Fig. 2a).
At day 4 after birth, the enamel-free cusps were easily detected both in the frontal section (Fig. 3) and in
the horizontal section (Fig. 4). Ameloblasts in the area
other than the enamel-free cusp were well differentiated and thick enamel was formed below them. However, ameloblasts in the enamel-free cusp were very
similar t o PALCs of 1-day-old rat; namely, they were
short and not polarized (Figs. 3b, 4b). In the enamelfree cusp hematoxylin positive enamel matrix-like material was not clearly detected, but in the marginal
region of the enamel-free cusp a thin layer of enamellike matrix was recognized and it continued into the
typical thick enamel matrix (Figs. 3b, 4b). Although
distinct enamel matrix was not observed in the
enamel-free cusps by H + E staining, almost continuous
immunostaining for amelogenin was distinctly detected at the interface between PALCs and dentin in
the enamel-free cusps both in the frontal section (Fig.
3a) and in the horizontal section (Fig. 4a). However,
the penetration of immunoreactive material toward
the dental pulp was not observed (Figs. 3a, 4a).
Control sections stained using PBA in place of En3
showed faint background staining in predentin (Fig.
5a).
enamel bearing region. The width of the layer varied
and it was often incomplete as shown in Figure 9. No
basement membrane was observed beneath PALCs
(Figs. 8,9).The Golgi apparatus of PALCs (Fig. 10) and
stippled material-like substance in the intercellular
space between PALCs (Fig. 10, inset) showed immunolabelling for amelogenin.
DISCUSSION
Non-enamel bearing regions of molar occlusal CUSPS
had been reported in the rat (Addison and Appleton,
1921; Mellanby, 1939; Lefkowiz et al., 1953) and in the
mouse (Gaunt, 1956; Cohn, 1957). These investigators
concluded that the cells of the inner enamel epithelium
of non-enamel bearing regions did not differentiate
into secretory ameloblasts, so enamel was not deposited on the dentin surface in the enamel-free cusp.
However, Johannessen (1961) showed the presence of
enamel on the enamel-free cusps of rat molars by microradiography. And Sutcliffe and Owens (1980) observed in their ultrastructural investigations that
some patches of enamel-like matrix were deposited on
the dentin surface in the enamel-free cusps of rat molars. Sutcliffe and Owens (1980, 1981) also indicated
that the cells of the inner enamel epithelium in the
enamel-free cusp secreted enamel-like matrix on the
dentin surface and subsequently they resorbed the
enamel-like matrix as well as the dentin surface just
prior to tooth eruption, so the enamel-free cusp actually lacked enamel. Similarly, Diab and Zaki (1985)
and Sakakura et al. (1989) reported the presence of an
afibrillar layer on the dentin surface in the enamel-free
cusp of mouse molar in light and electron microscopic
studies. However, it is not possible to conclude only on
the basis of its morphological feature that the afibrillar
extracellular matrix in the enamel-free cusp is enamel.
In the present immunohistochemical study using the
monoclonal antibody En3 against rat amelogenin, we
demonstrated that the Golgi apparatus of presecretory
ameloblast-like cells (PALCs) in the enamel-free cusp
showed positive staining and that the amorphous material on the dentin surface beneath PALCs showed
labelling by immunoelectron microscopy. These observations suggest that PALCs in the enamel-free cusp
differentiate into the cells which secrete the amorphous material containing amelogenin on the dentin
surface in the enamel-free cusp. This suggestion is consistent with the previous report by Nakamura et al.
lmmunoelectron Microscope Observations
(1986) at the light microscopic level that there was
Control sections incubated with PBL in place of En3 discrete immunostaining for mouse amelogenin at the
showed negligible labelling (Fig. 6).
interface between PALCs and adjacent dentin in the
In a large portion of the crown of the mandibular enamel-free cusps of mouse molars. The amorphous
first molar at day 4, the secretory ameloblasts with
Tomes’ processes and enamel matrix with typical demineralized enamel structure were observed as shown
in Figure 7. Enamel matrix showed heavy labelling for
the monoclonal antibody En3. On the other hand, in
Fig. 8 . The interface between presecretory ameloblast-like cells
the enamel-free cusp, PALCs without Tomes’ process (PALCs) and dentin (D) in the enamel-free cusp. In spite of lack of
processes in PALCs, a layer of amorphous material (AM)
were observed instead of secretory ameloblasts (Figs. 8, Tomes’
which shows positive labelling for En3 a t the interface between
9). At the interface between PALCs and dentin there PALCs and dentin is observed. There is no basement membrane bewas a layer of amorphous material showing positive neath these cells. X62,OOO. Inset shows some patches of AM with
labelling for En3 instead of typical enamel matrix (Fig. labelling for En3 in dentin in the enamel-free cusp. x 46,000.
8). Some patches of labelled material with various size
Fig. 9. The interface between presecretory ameloblast-like cells
and shape were observed in dentin or predentin matrix (PALCs) and dentin (D) in the enamel-free cusp where there is no
in the enamel-free cusp (Fig. 8, inset) as well as in the En3-positive material. x 34,000.
AMELOGENIN I N T H E ENAMEL-FREE CUSP
Figs. 8 and 9.
593
594
T. INAI ET AL.
Fig. 10. Golgi region of presecretory ameloblast-like cell in the enamel-free cusp. Note the labelling of
condensing vacuoles indicating the synthesis of amelogenin in these cells. X 48,000. The presence of
stippled material-like substance with labelling in the intercellular space between presecretory ameloblast-like cells is shown in inset. x 40,000.
dense material which resembled the amorphous material in the enamel-free cusp was described at the
dentino-enamel junction in papers by Warshawsky
(1971) and Warshawsky and Vugman (1977). Similar
material was decribed on the root-analogue surface of
rabbit incisors, formed by Hertwig’s sheath and identified as enamel-like by Schonfeld and Slavkin (1977).
Antibodies to amelogenin were localized between presecretory ameloblasts and dentin by Slavkin et al.
(1988a) and by Nanci et al. (1989). These authors suggested that presecretory ameloblasts in the areas other
than the enamel-free cusp also had the ability to synthesize and secrete the amorphous material containing
amelogenin on the dentin surface. These previous studies as well as data presented here support the view that
the material at the surface of dentin in the enamel-free
cusp is a natural secretion product that precedes normal amelogenesis and is formed by the presecretory
ameloblasts in preparation for enamel formation. The
same factors that prevent enamel from forming on the
surface of dentin in the roots might also prevent
enamel formation on the enamel-free cusps. Although
Sakakura et al. (1989)reported the differences between
the afibrillar matrix in the enamel-free cusp and
enamel in normal amelogenesis, the amorphous material in the present study is similar to enamel matrix
because it contains amelogenin, which is the enamel
matrix specific major protein, and it is the product derived from presecretory ameloblast-like cells in the
enamel-free cusp, but not from odontoblasts. It has
been unclear whether the amorphous material contains enamelin. The molecular constituents of the
amorphous material need to be investigated.
Recently, Inai et al. (1991) demonstrated using an
immunohistochemical technique that presecretory
ameloblasts secreted stippled material-like substance
containing amelogenin into the predentin and this substance penetrated into the dental pulp in rat molar
tooth germs. They hypothesized that the penetration of
amelogenin might play a role in the interaction between presecretory ameloblasts and odontoblasts. If
this hypothesis is true, the penetration of amelogenin
should occur also in the enamel-free cusp because ectomesenchymal cells in the enamel-free cusp differentiate into odontoblasts and sequential dentinogenesis
occurs normally.
It was very easy to distinguish the non-enamel bearing region in the enamel-free cusp from the area of
normal amelogenesis in the cusp of 4-day-old rat mandibular first molar because distinct thick enamel existed in the latter but no clear enamel was detected in
the former by light microscopy. However, it was difficult to detect the non-enamel bearing region in the
cusp of the 1-day-oldrat molar because clear hematoxylin positive enamel matrix had not been produced
even in the area other than the non-enamel bearing
region. In the present study, we regard “PALCs at the
cusp tip of 1-day-old rat mandibular first molar” as
ameloblasts in the future enamel-free cusp by the following two criteria. 1) The nuclei of most of amelo-
595
AMELOGENIN I N THE ENAMEL-FREE CUSP
ACKNOWLEDGMENTS
blasts in the cusp migrated to the proximal cytoplasm
and they were elongating, but PALCs at the cusp tip
This work was supported in part by a Grant for Scihad not polarized. 2) PALCs a t the cusp tip were close entific Research from the Japanese Ministry of Educato the outer enamel epithelium. Sutcliffe and Owens tion, Science and Culture (project 63771448).
(1980) also reported that this proximity was observed
at the cusp tips of rat mandibular first molars until
LITERATURE CITED
about 3 to 4 days after birth. We showed using l-dayAddison,
W.H.F.,
and
J.L.
Appleton 1921 On the development of the
old rat mandibular first molars that the penetration of
ameloblasts of the albino rat, with special reference to the
amelogenin from ameloblasts including PALCs in the
enamel-free area. Anat. Rec., 21:43 (Abstract).
future enamel-free cusp to the dental pulp also oc- Cohn, S.A. 1957 Development of the molar teeth in the albino mouse.
Am. J. Anat., 101:295-319.
curred before enamel formation. A thin layer of dentin
M.M., and A.E. Zaki 1985 Ultrastructure and lysosomal cytowas formed in the cusp of the 1-day-oldrat molar. This Diab,chemistry
of mouse molar enamel-free areas. J. Dent. Res., 64:
penetration was not observed in the enamel-free cusp
324 (Abstract).
of the 4-day-old rat molar, where thick dentin had been Gaunt,
W.A. 1956 The development of enamel and dentin on the mo.
lars of the mouse, with an account of the enamel-free areas. Acta
formed by odontoblasts. But some patches of amor28:lll-134.
phous material with labelling for amelogenin were rec- Inai,Anat.,
T., T. Kukita, Y. Ohsaki, K. Nagata, A. Kukita, and K. Kurisu
ognized in the dentin in the enamel-free cusp by im1991 Immunohistochemical demonstration of amelogenin penemunoelectron microscopy. These patches with labelling
tration toward the dental pulp in the early stages of ameloblast
develonment in rat molar tooth eerms. Anat. Rec.. 229:259-270.
in the dentin in the enamel-free cusp may imply that
L.B. 1961 Presence of enamel-covered cusps in rat mothe penetration of amelogenin had occurred in the Johannessen,
lars. Archs Oral Biol., 5:61-62.
enamel-free cusp, because odontoblasts do not show Johnson, G.D., and G.M.C.N. Araujo 1981 A simple method of reducamelogenin gene expression (Snead et al., 1988).These
ing the fading of immunofluorescence during microscopy. J . Immunol. Methods, 43:349-350.
investigations indicate that the penetration of ameloE. 1971 Electron microscopy of the differentiating rat
genin occurs in the enamel-free cusp as well as in the Kallenbach,
incisor ameloblast. J . Ultrastruct. Res., 35.508-531.
area of normal amelogenesis (Inai et al., 1991), and Karnovsky,
M.J. 1971 Use of ferrocyanide reduced osmium tetroxide
support the hypothesis that the penetration of ameloin electron microscop . Proceedings of the American Society on
Cell Biology, New Orreans, Louisiana, 281:146 (Abstract).
genin might play a role in the interaction between preE.J. 1972 Histogenetic aspects of dermal-epidermal interacsecretory ameloblasts including PALCs and odonto- Kollar,
tions. In: Developmental Aspects of Oral Biology. H.C. Slavkin
blasts.
and L.C. Bavetta, eds. Academic Press, New York, pp. 126-149.
However, the exact role of the penetration of amelo- Kollar, E.J. 1983 Epithelial-mesenchymal interactions in the mammalian integument: Tooth development as a model for instructive
genin as well as the amorphous material in predentid
induction. In: Epithelial-Mesenchymal Interactions in Developdentin is unknown. The amorphous material may play
ment. R.H. Sawyer and J.F. Fallon, eds. Praeger, New York, pp.
a role in the epithelial-mesenchymal interactions oc27-50.
curring during odontogenesis and/or may participate in Lefkowitz, W., C.F. Bodecker, and D.F. Mardfin 1953 Odontogenesis
of the rat molar: Prenatal stage. J . Dent. Res., 32:749-772.
the initial mineralization of the dentin extracellular
I.W., and P.K. Nakane 1974 Periodate-lysine-paraformalmatrix and the formation of the dentino-enamel junc- McLean,
dehyde fixative, a new fixative for immunoelectron microscopy. J.
tion (Slavkin et al., 1988a; Warshawsky, 1988; Nanci
Histochem. Cytochem., 22:1077-1083.
et al., 1989). The penetration of amelogenin first ap- Mellanby, H. 1939 The development of teeth in the albino rat. Br.
Dent. J., 66:76-86.
peared in differentiation I1 of Kallenbach (19711, in
which the basement membrane between presecretory Nakamura, M., P. Bringas, and H.C. Slavkin 1986 Immunohistochemical comparisons of region-specific dental epithelial-derived
ameloblasts and odontoblasts was intact and smooth,
ECM proteins. Anat. Rec., 214:90A (Abstract).
and almost disappeared a t initial enamel formation, Nakamura, M., P. Bringas, and H.C. Slavkin 1987 Position characand that during this period, odontoblasts endocytosed a
teristics of “Intercellular communication” during odontogenetic
epithelial differentiation. Anat. Rec., 218t96A (Abstract).
part of the stippled material-like substance containing
A,, J.P. Ahluwalia, J.R. Pompura, and C.E. Smith 1989 Bioamelogenin which penetrated from presecretory amelo- Nanci,
synthesis and secretion of enamel proteins in the rat incisor.
blasts into the dental pulp (Inai et al., 1991). In differAnat. Rec., 224:277-291.
entiation 11-111, odontoblasts first showed positive im- Ruch, J.V. 1985 Epithelial-mesenchymal interactions in formation of
mineralized tissues. In: The Chemistry and Biology of Mineralmunostaining with anti-mouse dentin phosphoprotein
ized Tissues. W.T. Butler, ed. Ebsco Media, Inc., Birmingham,
(DPP) polyclonal antibodies in their cytoplasms and
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Y., N. Fujiwara, and T. Nawa 1989 Epithelial cytodifferperiod of the penetration of amelogenin. Therefore, the Sakakura,
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mesenchymal interactions and/or the initiation of min- Slavkin, H.C., M. MacDougall, M. Zeichner-David, P. Oliver, M. Nakamura, and M.L. Snead 1988b Molecular determinants of craeralization of predentin.
~~~
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cusp, immunoelectron, free, molar, microscopy, germsimmunofluorescent, demonstration, enamel, toots, rat, studies, amelogenin
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