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Immunohistochemical demonstration of amelogenin penetration toward the dental pulp in the early stages of ameloblast development in rat molar tooth germs.

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THE ANATOMICAL RECORD 229:259-270 (1991)
Immunohistochemical Demonstration of
Amelogenin Penetration Toward the Dental Pulp
in the Early Stages of Ameloblast Development in
Rat Molar Tooth Germs
TETSUICHIRO INAI, TOSHIO KUKITA, YASUYOSHI OHSAKI, KENGO NAGATA,
AKIKO KUKITA, AND KOJIRO KURISU
Second Department of Conservative Dentistry (T.I.),and Second Department of Anatomy
(T.K., Y.O., K.N., A.K., K.K.), Faculty of Dentistry, Kyushu University, Fukuoka, Japan
ABSTRACT
In order to examine the synthesis and secretion of enamel protein
by ameloblasts in their early stages of development, immunohistochemical localization was carried out a t light and electron microscopic levels using a monoclonal
antibody produced in a preliminary experiment. Materials used were tooth germs
of mandibular first molars of rats a t 0-5 days after birth.
Immunoblot analysis after two-dimensional electrophoresis revealed that antigen molecules recognized by the monoclonal antibody were amelogenins of 26-28
kDa (PI, 6.6-7.0).
An immunohistochemical examination using this monoclonal antibody demonstrated that the presecretory ameloblasts in their early stages of differentiation
both synthesized amelogenin and secreted through a classical merocrine secretory
pathway. In some presecretory ameloblasts as well as ameloblasts we observed the
distended cisternae of rough endoplasmic reticulum (rER) which demonstrated
heterogenous immunolabelling. The immunolabellings were also detected in the
predentin a s well a s the intercellular spaces of odontoblasts and dental pulp cells
which indicated penetration of amelogenin from the presecretory ameloblast layer
to the dental pulp. The presence of coated pits a t the plasma membrane of odontoblasts in close proximity to enamel protein along with the immunolabelling of
lysosomes of the odontoblasts suggests the phagocytosis of the enamel protein into
the odontoblasts. These observations suggest the possibility that the penetration of
enamel protein toward the dental pulp and odontoblasts plays a role in the interaction between ameloblasts and odontoblasts.
The developing enamel matrix contains a complex
mixture of proteins. It is, therefore, essential to clearly
elucidate the nature of these proteins in order to reach
a better understanding of amelogenesis. The enamel
proteins secreted by ameloblasts can be divided into
two classes, amelogenins and enamelins, based on their
solubility properties, amino acid composition, molecular weight, and isoelectric point (Termine et al., 1980;
Belcourt e t al., 1983; Rosenbloom et al., 1986; Ogata et
al., 1988). Amelogenins are hydrophobic and prolinerich proteins with a relatively low molecular weight
ranging from 6 to 27 kDa, whereas enamelins are hydrophilic glycoproteins with a relatively high molecular weight ranging from 8 to 72 kDa. Newly secreted
enamel proteins are deposited into the enamel matrix
and are rapidly reduced to less than 2% of the original
amount during the process of enamel maturation.
The immunohistochemical localization of enamel
proteins in tooth germs has been previously described
a t the light microscopic (Graver et al., 1978; Slavkin et
al., 1981,1982)and electron microscopic level (Nanci et
al., 1984, 1985,1987,1989;Herold et al., 1987; Slavkin
et al., 1988; Inage e t al., 1989).Recently some investic 1991 WILEY-LISS.
INC
gators (Slavkin et al., 1988; Nanci et al., 1989; Inage et
al., 1989) have reported that presecretory ameloblasts
synthesize and secrete enamel proteins into predentin
prior to or after the breakdown of the basement membrane. However, the exact role of these “ectopic”
enamel proteins is still unclear.
In the present study, we describe the detailed immunohistochemical localization of amelogenin in the early
stages of ameloblast differentiation using the monoclonal antibody against amelogenin obtained in a preliminary study. Immunolabelling was detected in the
presecretory ameloblasts both intra- and intercellularly as well as in the predentin, intercellular spaces of
the odontoblasts and the dental pulp cells. These results suggest the penetration of amelogenin from the
presecretory ameloblast layer toward the dental pulp.
Received May 7, 1990; accepted J u l y 24, 1990.
Address reprint requests to K. Kurisu, Second Department of‘ Anatomy, Faculty of Dentistry, Kyushu University 63, Maidashi. Higashiku, Fukuoka 812, Japan.
260
T.INAI ET A L
MATERIALS AND METHODS
preparation of Antibodies
lmmunoblot Analysis
Protein extraction
One group of samples was prepared according to the
Preparation of irnmunogen
method of Zeichner-David et al. (1988) with some modIncisor tooth germs with some surrounding tissues ifications. Mandibular incisors were dissected from 7
were dissected from 3-6 week-old rats in 0.1 M phos- day-old rats and washed with ice-cold PBS. They were
phate-buffered saline (PBS). Apical ends of the tooth homogenized with Polytron (Kinematica, Luzern, Switgerms containing differentiating, presecretory, and zerland) in ice-cold 0.5 M acetic acid and then extracted
secretory ameloblasts were cut away and homogenized with 0.5 M acetic acid using a dialysis bag (3,500 Da
with tef lon-glass homogenizer in ice-cold Iscove’s mod- cut-off) for 7 days a t 4°C. The extract was centrifuged
ified Dulbecco’s medium (IMDM; Gibco, Grand Island, a t 13,000g for 10 minutes a t 4°C and the supernatant
NY) containing 0.5% sodium dodecylsulfate.
was lyophilized. Other groups of samples were prepared
according to the method of Termine e t al. (1980).
In vitro immunization and production of hybridornas
Unerupted fetal bovine molars were dissected and the
The in vitro immunization was performed a s de- enamel surfaces were wiped off to remove any tissue
scribed by Reading (1982) and Boss (1984) with some covering their surfaces. The scratchable enamel (cheesy
modifications. Briefly, a single cell suspension was pre- enamel) was serially extracted with 4 M guanidine HCl
pared from spleens which were removed aseptically (“G’ extract) and then with 4 M guanidine HC1 confrom 6-8 week-old unimmunized female Balbic mice. taining 0.5 M ethylenediamine tetraacetic acid (EDTA)
After washing and centrifugation, the spleen cells in (“E” extract). All procedures were performed at 4°C in
IMDM (supplemented with 5 x
M 2-mercapto- the presence of protease inhibitors. The extracts were
ethanol, 100 unitdm1 penicillin, and 100 pgiml strep- concentrated using dialysis bags which were aspirated
tomycin) containing 20% fetal bovine serum (FBS; in flasks. They were then dialyzed against distilled
Whittaker Co., Walkersville, MD) were incubated for 4 water a t 4”C, and then freeze-dried.
days in a CO, incubator in the presence of 1-100 pl Electrophoresis and imrnunodetection
tooth germ homogenate and 20 pgiml N-acetylmuSingle-dimensional sodium dodecylsulfate polyacrylramyl-L-alanyl-D-isoglutamine (Peptide Institute Inc.,
Minoo, Japan) as adjuvant. The immune spleen cells amide gel electrophoresis (SDS-PAGE) was carried out
and mouse myeloma cells (P3-X63-Ag8-U1) were fused according to the Laemmli procedure (1970) using
a t a ratio of 1O:l in 0.5 ml solution containing 50% 12.5% acrylamide gel under both reducing and nonrepolyethylene glycol 4000 and 5% dimethyl sulfoxide in ducing conditions. The stacking gel contained 4% polydouble distilled water. The fused cells were adjusted to acrylamide while the separating gel contained 12.5%
1 x lo6 spleen cellsiml in HAT medium (IMDM sup- polyacrylamide. SDS-PAGE Standards (Low range; Bio
plemented with hypoxanthine, aminopterin, and thy- Rad Lab., Richmond, CA) were used for molecular
midine) containing 20% FBS and plated into 24-well weight standards. After electrophoresis, electroblotplates (Falcon, Lincoln Park, NJ). The fused cells were ting of proteins onto nitrocellulose membrane (Transfed in HAT medium for 10-14 days by exchanging 0.5 Blot; Bio Rad Lab.) was done using TEFCO blot equipment (TEF Co., Machida, Japan) as described by
ml of the medium on every third day.
Towbin et al. (1979). The gel was transferred a t 180
mA for 1 hour in a buffer containing 25 mM Tris, 192
Antibody screening and hybridorna cloning
mM glycine, and 20% methanol.
The culture media from the wells containing macroTwo-dimensional SDS-PAGE was conducted by the
scopically visible colonies were screened by indirect im- method described by O’Farrell (1975). The first dimenmunofluorescence using paraffin sections of rat jaws sion employed 2% Ampholine (pH 3.5-10; Pharmacia
containing tooth germs. The details are described in LKB Biotechnology, Piscataway, NJ) in a 2.5 x 130
the section on immunofluorescence procedure. The hy- mm tube and was focused for 1 hour a t 200 V, 15 hours
bridomas producing the antibodies which react with at 300 V, and then 1 hour a t 500 V. SDS-PAGE Stanspecific cells or extracellular components in tissue sec- dards were used for the molecular Standard. The septions were cloned four times by limiting dilution.
arated proteins were transferred to nitrocellulose
In the present study the monoclonal antibody En3 membranes as previously described (Towbin et al.,
(Inai, unpublished data), which reacted with amelo- 1979).
blasts and enamel matrix, is used.
Both in single- and two-dimensional immunoblot
analyses, some of the nitrocellulose membranes were
Production of polyclonal anti-laminin antibody
stained with 0.1% amido black 1OB to detect the transPolyclonal anti-laminin antibody was prepared a s ferred proteins. Others were incubated first with monofollows: laminin was extracted from EHS sarcoma tis- clonal antibody En3 and second with horseradish persue and purified by chromatography on DEAE-cellu- oxidase-conjugated goat anti-mouse p chain antibody
lose and Agarose A15 and A5 column, a s described by (Zymed Lab. Inc., South San Francisco, CA) according
Timpl et al. (1979). The mouse tissue of EHS sarcoma to the methods of Slavkin et al. (1988) to detect the
was generously donated by K. Kimata, Faculty of Sci- antigen.
ence, Nagoya University, Japan. The antibody to lamiImmunofluorescence Procedure
nin was raised in rabbits and then affinity-purified
The 0-5 day-old rats were perfused with Periodatewith Sepharose-4B column, a s described previously
Lysine-paraformaldehyde fixative (McLean and Na(Linsenmayer, 1981; Ruoslahti et al., 1981).
26 1
ENAMEL PROTEIN IN DENTAL PULP
kDa
9 76 64 3-
3 1-
2 2-
Fig. 1. Immunoblot analysis of acetic acid extract of rat incisor tooth
germs separated by one-dimensional SDS-PAGE. The samples were
fractionated by gel electrophoresis under reducing conditions, transferred to nitrocellulose paper, and then incubated with En3. The antigen-antibody complex was identified by an enzymatic colorimetric
reaction using horseradish peroxidase. Lane 1 was stained with
amido black. Lane 2 was a n immunoblot. A single band of approximately 23 kDa is detected with En3 in lane 2.
14-
8
kDa
Fig. 2. Immunoblot analysis of " G extract of bovine enamel protein
separated by two-dimensional SDS-PAGE. The samples were run in
tube isoelectric focusing gels, separated by SDS-PAGE under reducing conditions, and then transferred to nitrocellulose paper. (a)Amido
black stain; (b)immunoblot using En3. A single spot with a molecular
mass of 26-28 kDa and PI of 6.6-7.0 is detected with En3 in b.
2
1
I
7
I
6
I
5
I
4 pH
I
8
I
7
I
6
I
5
I
4pH
I
9 76 64 3-
3 1-
2 214kane, 1974) through the left ventricle and the dissected
mandibles were immersed in the same fixative for 3-5
hours at 4°C. After decalcification by 10% EDTA for
1-3 weeks at 4"C, the specimens were embedded in
paraffin. Sections mounted on glass slides were incu-
bated with either the hybridoma medium under examination or with monoclonal antibody En3 for 1 hour a t
room temperature. Control sections were treated with
either normal mouse serum (Zymed Lab. Inc.) or PBA
(PBS containing 1%chick albumin) instead of mono-
262
T. INAI ET AI,
Figs 3-5
ENAMEL PROTEIN I N DENTAL I ’ U I 2
clonal antibody. After repeated rinsing with PBS, the
tissue sections were incubated with fluorescein isothiocyanate (FITC) labelled goat anti-mouse immunoglobulin (Ig) or FITC labelled goat anti-mouse p. chain antibody (Zymed Lab. Inc.) for 30 minutes a t 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 a s described by Johnson and Araujo (1981). The
specimens were then examined by a Zeiss fluorescence
microscope.
26:
body. 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
2OOCX electron microscope.
RESULTS
Characterization of Antigen of Monoclonal Antibody En3
Figure 1 shows the SDS-PAGE pattern and immunoblot analysis of acetic acid extract of the homogenate
The 0-5 day-old rats were perfused through the left of rat incisor tooth germs. Since the preliminary imventricle with a mixture of 1%glutaraldehyde and 3% munohistochemical study demonstrated that En3
paraformaldehyde in 0.1 M cacodylate buffer contain- cross-reacted with the bovine enamel matrix, the saming 0.05% CaCl,, pH 7.4. The mandibles were dissected ples from the bovine enamel matrix, which were easy
and immersed in the same fixative for 3-5 hours a t to get in large amounts, were also examined. Using
4°C. After decalcification in 10% EDTA for 1-3 weeks En3 a positive band was detected a t approximately 23
at 4”C, the mandibular first molars were cut with a kDa in the acetic acid extract under reducing condition
Microslicer into thick sections of 200-300 p. which (Fig. 1).Similar result was obtained under nonreducwere postfixed in osmium tetroxide reduced with po- ing conditions (data not shown).
Two-dimensional immunoblot analysis on bovine
tassium ferrocyanide (Karnovsky, 1971) for 2 hours a t
room temperature. The thick sections were washed in a “G’ extract revealed protein with a molecular weight of
sodium cacodylate buffer, dehydrated in a graded se- 26-28 kDa (PI, 6.6-7.0) using the En3 (Fig. 2). No
ries of ethanol, and embedded in Epon 812. Ultrathin reaction was detected when two-dimensional immunosections were mounted on nickel grids and floated on blot analysis on bovine “E” extract using En3 was condrops of a saturated aqueous solution of sodium ducted (data not shown). These results definitely sugmetaperiodate for 1hour and on PBL (0.1 M PBS con- gest that En3 specifically binds with amelogenin but
taining 1 % bovine serum albumin (BSA) and 0.1 M not with other proteins including enamelin. FITC lalysine-HC1) for 30 minutes. They were then treated belled goat anti-mouse p. chain antibody reacted
with En3 antibody for 1 hour a t room temperature. strongly with tissue sections of tooth germs binding
Control sections were treated with either normal En3, indicating the Ig class of En3 being IgM (data not
mouse serum or PBL instead of En3. After repeated shown).
rinsing with PBS, the sections were incubated with
lrnrnunofluorescentLocalization of the
colloidal gold labelled goat anti-mouse IJ- chain antiEnamel Protein Antigen
lrnrnunoelectron Microscopy
The first appearance of positive staining for monoclonal antibody En3 was detected at the distal end of
the early stage presecretory ameloblast (Fig. 3a). The
intensity of staining in the presecretory ameloblast
layer increased with the development of the presecreFig. 3. Micrographs of early stage presecretory amelohlasts ( P A ) tory ameloblast. In order to examine the relation beand odontoblasts (Od) stained with En3 (a) and H-E (h).To examine tween the staining pattern with En3 and the basement
the basement membrane, an adjacent section was stained with anti- membrane, the adjacent section was stained with antilaminin antibody ( e ) .The first appearance of positive staining for En3
laminin antibody (Fig. 3c). The results demonstrated
(large arrow) is detected in the presecretory ameloblast layer along
that the first appearance of staining for En3 was obthe basement membrane prior to its breakdown (arrowheads),and the
intensity of staining in the presecretory ameloblast layer increases
served prior to the breakdown of the basement memwith the development of the presecretory ameloblasts. Note the presbrane and that there was positive staining in the
ence of positive staining in the odontoblast layer (small arrows) prior
odontoblast layer prior to and after the breakdown of
to or after the breakdown of the basement membrane, although the
the basement membrane, although the intensity of the
intensity of staining increases after the breakdown o f t h e basement
membrane. a x : x 290.
staining increased after its breakdown. Along with the
development of the presecretory ameloblasts, heavy
Fig. 4. Micrographs of the late stage of presecretory ameloblasts
staining was also detected in the predentin and odon(PA) stained with En3 (a) and H-E (b). Heavy staining for En3 is
observed in the predentin (large arrow) and odontoblast layer (Od) toblast layer (Fig. 4). Retention of immunoreactive ma(small arrow). The staining in the odontoblast layer markedly de- terials in the subodontoblastic zone was detected at the
creases after the initiation of enamel matrix deposition (large arrow- pulp horn (Fig. 5). Accompanying the enamel matrix
head in b). Spot-like staining detected in the predentin seems to cordeposition, the staining in the odontoblast layer was
respond to hematoxylin positive material (small arrowheads in a and
also found to decrease markedly, and spot-like staining
h). a,b: x340.
was detected in the predentin (Fig. 4).
Fig. 5. Micrographs of the late stage of presecretory ameloblasts at
Control sections stained using normal mouse serum
the crest of a cusp stained with En3 (a)and H-E (h).Note the positive
or
PBA in place of En3 showed either negative or very
staining a t the subodontoblastic zone of the dental pulp (arrow).a.b:
faint background staining (data not shown).
x 290.
Figs. 3-5. Sections of mandibular first molars of 2 day-old rats.
After recording of the immunofluorescent reaction using En3 the
same sections were stained with hematoxylin and eosin (H-E) for
histological observations.
264
rr. I N A I Err XI,
ENAMEL PROTEIN IN DENTAL PULP
265
Lysosomes with labelling were often seen in the odontoblasts (Fig. 12). The presence of labelled SM in the
After applying En3 to the ultrathin sections, a spe- dental pulp continued until initial enamel formation.
At the late stage of presecretory ameloblasts, correcific labelling was first detected in the early stage of
sponding
to Differentiation V in which the basement
the presecretory ameloblasts corresponding to the Differentiation I1 of Kellenbach (1971),in which the base- membrane cannot be recognized, the labelled SM were
ment membrane between presecretory ameloblasts and observed in the intercellular spaces of the stratum inodontoblasts is uninterrupted and smooth. Gold parti- termedium and the proximal end of the presecretory
cles were present particularly over the Golgi appara- ameloblasts (Fig. 14). The distal surface of the pretus, rough-surfaced endoplasmic reticulum (rER),pale secretory ameloblasts a t this stage was rough and the
or dark staining lysosome-like granules, and the mul- basement membrane could not be recognized (Fig. 15).
tivesicular bodies distributed throughout the supranu- Numerous patches of labelled SM were observed (Fig.
clear compartment (Figs. 6, 7). However, the intensity 15).
At the very late stage of presecretory ameloblasts,
of labelling over these large granules was variable. Lacorresponding
to Differentiation VI in which the first
belling was also observed over an irregular mass of the
granular material in the intercellular space (Fig. 6). In mineral deposits in the predentin occur, heavy deposits
this paper we call the granular material “stippled ma- of SM, of so called fine-textured materials with labelterial-like substance” (SM). In this stage, the distal end ling, were detected a t the distal end of the cells (Fig.
of the presecretory ameloblast was smooth and the 16).
At the early stage of secretory ameloblasts, secretory
basement membrane was intact (Fig. 8 ) . Labelled SM
vesicles
in Tomes’ processes and enamel matrix were
was also observed in the intercellular space between
the presecretory ameloblasts and odontoblasts (Fig. 8). labelled (Fig. 17). In the dentin matrix a t this stage,
In the middle stage of the presecretory ameloblasts labelled patch-like materials of various sizes were obcorresponding to Differentiation 111-IV of Kallenbach served (inset of Fig. 17).
Control sections incubated with PBL in the place of
(Differentiation 111, the basement membrane is smooth
and uninterrupted except for penetrating filaments. A En3 showed a very small amount of background labelfew small droplets of coarse-textured material are ling (Fig. 18).
present in the predentin; Differentiation IV, the preDISCUSSION
secretory ameloblasts send processes of varying size
The
present
a
s
well
a s previous results (Nanci et al.,
and shape through the basement membrane into the
1985,
1989;
Inage
et
al.,
1989) suggest that the secrepredentin), distended rERs containing moderately
dense material with labelling were often observed, tion products of both presecretory ameloblasts and
while no labelling was detected in those containing ameloblasts follow a classical merocrine secretory
pale material (Fig. 9). These structures were observed pathway through the rER, the Golgi apparatus, the
either in the supranuclear compartment or in the in- secretory granules, and release at the cell surface. Imfranuclear compartment. At this stage numerous munolabelling with anti-amelogenin antibody En3 remasses of SM with labelling were detected in the pre- veals antigenicity in all these compartments. The fact
dentin (Fig. lo), and in the intercellular spaces of both that the rER is immunoreactive indicates that the anthe odontoblasts (Figs. 11, 12) and the dental pulp su- tigenic domain of amelogenin is already defined a s the
bodontoblastic zone (Fig. 13). Coated pits were ob- proteins are translated (Nanci et al., 1984, 1985).
The labelling for amelogenin was first detected a t
served on the lateral plasma membrane of the odontoblasts in contact with the labelled SM (Fig. 11). the presecretory ameloblasts intracellularly a s well a s
extracellularly in the early stages of presecretory
ameloblasts corresponding to Kallenbach’s Differentiation I1 (Kallenbach, 1971) in which the basement
membrane was intact. This finding is consistent with
those
of Nanci et al. (1989) and Inage et al. (1989).
Figs. 6-1 8. Immunoelectron microgaphs of mandibular first molars
The burst of amelogenin penetration toward the denof 1-2 day-old rats. Ultrathin sections were incubated wtih En3 or
PBL for control, and then incubated with colloidal gold labelled goat tal pulp was prominently detected after a breakdown of
anti-mouse p chain antibody.
the basement membrane (Differentiation IV). This
penetration
decreased accompanying the dentin minFig. 6. Supranuclear zone of the early stage presecretory ameloeralization and almost disappeared when the initial
blasts. Golgi apparatus (GI, dark type lysosomes (dLy) and stippled
enamel was formed. Although immunohistochemical
material-like substance (SM) are labelled. x 31,700.
localizations of enamel proteins in tooth germs have
Fig. 7 . Supranuclear zone of the early presecretory ameloblasts.
Dark type lysosomes tdLy) a s well a s pale type lysosomes (inset,pLy1 been reported by many researchers a t the light microscopic level (Graver et al., 1978; Slavkin et al., 1981,
are labelled. x 24,900. Inset, x 18,000.
1982) as well as a t the electron microscopic level
Fig. 8. Distal end of the early stage presecretory ameloblasts (PA). (Nanci et al., 1984, 1985, 1987, 1989; Herold e t al.,
Dark type lysosomes (large arrow) and stippled material-like sub1987; Slavkin e t al., 1988; Inage et al., 1989), the posstance (small arrow) in intercellular space (ICS)between presecretory
itive immunolabelling for the enamel proteins has not
ameloblasts and odontoblasts are labelled. Note the presence of a n
yet been described in the dental pulp. One of the reaintact basement membrane. x 39,600.
sons why previous authors failed to investigate this lies
Flg. 9. Infranuclear compartment of the middle stage presecretory
in the difference of the objectives of our respective studameloblast ( P A ) .Distended rERs (dER) containing moderately dense
ies.
Another reason may be the difference of antibodies
materials show labelling, while no labelling is observed in that conused. The previous researchers, except for Herold et al.
taining pale material (PER,. x 37,900.
lmmunoelectron Microscopic Localization of the Enamel
Protein Antigen
266:
T.I N A I ET AI,
ENAMEL, I’KOTEIN I N I)ENTAI, I’LJLP
(1987), used polyclonal antibodies, while we used a
monoclonal antibody in the present study. Polyclonal
antibodies against a molecule are a mixture of monoclonal antibodies against several antigenic determinants of the molecule. Therefore, it is believed that, in
immunohistochemistry, polyclonal antibodies generally show more intensive staining than monoclonal antibodies. However, the reverse is true when the antigenic determinant of a molecule for the monoclonal
antibody is dominant in a tissue although polyclonal
antibodies contain very few antibodies against the determinant (Yasuda e t al., 1988).The antigenicity of the
enamel protein may be reduced by the modification or
degradation of antigenic determinants during the process of penetration into the dental pulp, which induces
a decrease in immunoreactivity of the polyclonal antibodies against the protein. However, the antigenic determinant of the monoclonal antibody En3 is conserved
during the process of penetration into the dental pulp.
This may be one of the reasons why the previous investigators using polyclonal antibodies have not detected the penetration of enamel proteins into the dental pulp.
Reith (1967) reported that the enamel precursor-like
material referred to as stippled material was observed
in predentin, on the surface of the enamel matrix, and
between ameloblasts in rat molar tooth germs. On the
other hand, Kallenbach (1971) found several types of
extracellular dense material differing from each other
by texture and time of appearance in rat incisors.
Coarse-textured material appeared first between presecretory ameloblasts, then within predentin when the
basement membrane was perfectly intact. Fine-textured material appeared, after .the disappearance of the
basement membrane, in the same position within the
predentin formerly occupied by coarse-textured material. These materials, however, have often been detected also in calcified dentin (Suga, 1960; Watson,
1960; Reith, 1967) or deep within predentin (Kallenbach, 1971) in both r a t incisors and molars. Yamamoto
et al. (1980) have also observed the SM in dentin and
deep in predentin a s well as between odontoblasts. In
the present paper, we call the SM present in the area
Fig. 10. Predentin (PDI, distal end of odontoblast (Odl, and cytoplasmic process of odontoblast (PI facing the middle stage presecretory ameloblasts in which the basement membrane is penetrated by
fine filaments or cytoplasmic processes of presecretory ameloblasts.
Stippled material-like substance (SMI deep in predentin is labelled.
x 51,000.
Fig. 11. Odontoblasts tOd1 and the intercellular space a t the middle
stage of presecretory ameloblasts. Stippled material-like substance in
the intercellular space is labelled. Note the presence of coated pits
(arrows) on the lateral plasma membrane of the odontoblasts.
x 41,300.
Fig. 12. Odontoblasts (Odl and the intercellular space a t the middle
stage of presecretory ameloblasts. Note the presence of labelling on
stippled material-like substance tSM) in the intercellular space and
the lysosome (Lyl. x 42,900.
Fig. 13. The intercellular space at the proximal end of odontoblasts
and cells of the dental pulp ( P u ) a t the middle stage of presecretory
ameloblasts. Stippled material-like substance tSM) in the space is
labelled. x 32,400.
267
other than the enamel matrix the “ectopic” stippled
material (ESM).
The origin of ESM is controversial. Reith (1967) and
Yamamoto et al. (1980) regarded ESM a s products of
ameloblasts because of the structural resemblance of it
to the enamel matrix before calcification. Kallenbach
(1971), however, doubts that ESM is of ameloblast-origin, because 1) ESM were detected in the predentin
layer while the basement membrane was still perfectly
intact, 2) they were never in contact with the basement
membrane but at varying distances away from it, and
3) many ESM were in close proximity to, or even in
contact with, the odontoblast processes. Our immunohistochemical examination a s well a s previous studies
(Slavkin e t al., 1988; Nanci et al., 1989; Inage et al.,
1989) revealed that ESM contain enamel protein. And
Snead et al. (1988)demonstrated that only ameloblasts
showed amelogenin gene expression while odontoblasts
did not. The basement membrane cannot be a perfect
barrier even for large molecules. These data support
the view that ESM is of ameloblast-origin.
The role of ESM is still obscure. ESM may play a role
in the initial mineralization of dentin (Slavkin e t al.,
1988; Inage et al., 1989). In our decalcified sections, the
concomitant absence of “crystal ghosts” (Bonucci, 1969)
or “electron lucent clefts” (Warshawsky and Vugman,
1977) in ESM, which would have suggested a trace of
crystal formation in the decalcified tissue, suggests
that crystals had not yet appeared in ESM. We observed coated pits on the lateral plasma membrane of
the odontoblasts in close proximity to ESM with labelling and labelled lysosomes in them. These observations suggest that the odontoblasts endocytosed the
ESM and processed it in lysosomes. The penetration of
ESM into the dental pulp occurred at a restricted developmental period, namely that it started before dentin mineralization, even before the breakdown of the
basement membrane and then disappeared a t initial
enamel formation. Inage et al. (1989) supposed that
ESM might have a n effect on the differentiation of
odontoblasts since these seemed to start the secretion
of phosphoproteins a t this stage. These observations
suggest the possibility that the penetration of ESM
into the dental pulp plays a role in the interaction between presecretory ameloblasts and odontoblasts.
However, there is a possibility that this penetration
might be the result of physical diffusion without having a specific biological role and might be blocked by
the presence of the layer of mineralized dentin. Further
investigation is needed to clarify the role and the fate
of ESM.
Nanci and Warshawsky (1984), and Uchida (1988)
reported that the stippled material appearing in the
secretory stage of ameloblasts was a n artifact depending on the fixation method. Whatever the true structure of the material is, it is considered to be a secretory
product of either the presecretory ameloblasts or
ameloblasts and to contain amelogenin.
Moe (1971)described the distended cisternae of rER
in some presecretory ameloblasts and ameloblasts.
Kallenbach ( 1972) detected spherical granules of electron dense materials within the distended cisternae of
rER and supposed that these granules were caused by
the accumulation of enamel proteins. The present immunohistochemical examination revealed immunola
268
T. I N A I KT AIL
Figs. 14-17.
269
ENAMEL PROTEIN IN DENTAL PU1,I’
nor differences between them. Although there were
clear circular gaps between the highly electron dense
granules and walls of cisternae in the figures of Kallenbach (1972), we observed homogenous and moderately electron dense material in the distended cisternae without clear gaps. This discrepancy of the
structure may come from differences of tissue preparation. In the present study, the immunolabelling of distended cisternae was heterogenous, namely some distended rERs were positive but other ones were
negative. In our previous study on the immunocytochemical localization of type I collagen in cultured
mesenchymal cells from palatal shelves of mouse fetuses, immunolabelling of the distended cisternae was
similarly heterogenous (unpublished data). Referring
to the experiments of Weinstock (19701, Kallenbach
(1972) supposed that the formation of intracisternal
granules was caused by a n interruption in the transport of newly synthesized protein from the rER to the
Golgi apparatus. If his hypothesis is true, the heterogenous distribution of immunolabelling detected in both
our previous as well a s present study may suggest the
presence of a sorting system in rER before the transport of newly synthesized proteins from the rER to the
Golgi apparatus. Therefore, the relationship between
the heterogenous immunolabelling and the phenomena
occurring in the cell needs to be investigated further.
ACKNOWLEDGMENTS
Fig. 18. Control section, incubated with PBL instead of En3, of the
odontoblast (Od) at the middle stage of presecretory ameloblasts and
stippled material-like substance (SM)in the predentin (PD). No specific labelling and very little background labelling are observed.
x 27.000.
belling for amelogenin over some distended cisternae,
supporting Kallenbach’s suggestion. The morphology
of distended cisternae with labelling are very similar
to that shown by Kallenbach (1972), but there are mi-
Fig. 14. Cells of stratum intermedium (SI)and proximal end of the
late stage presecretory ameloblasts (PA).Stippled material-like substance (SM) in the intercellular space is labelled. x 26,800.
Fig. 15. Distal end of the late stage presecretory amelohlasts (PA).
Stippled material-like substance or fine-textured material (arrows)in
contact with presecretory amelohlasts or in predentin (PD) are labelled. Note the indentation of the distal surface of the presecretory
ameloblasts and the breakdown of the basement membrane. Some
stippled material-like substance is in contact with the cytoplasmic
process (Pi of the odontoblast. x 20,000
Fig. 16 The distal end of the late stage presecretory amelohlasts
(PA).Stippled material-like substance (large arrow) in the invaginations of the distal cell membrane and fine-textured material (small
arrow), which appears more finely granular than stippled materiallike substance, in contact with the distal surface membrane of presecretory ameloblasts and in the dentin (D) are labelled. Secretory
granules (arrowheads) in the presecretory ameloblasts are also labelled. x 26,400.
Fig. 17. Distal end of the early secretory ameloblasts. Secretory
granules (arrows) in Tomes’ processes (TP) and enamel matrix (EM)
are labelled. x 17,700. Inset shows stippled material-like substance
in the dentin matrix a t this stage which reveals labelling. x 15,700.
We thank Dr. K. Kimata, Faculty of Science, Nagoya
University, for his gift of tissue masses of EHS sarcoma. We also thank Mr. B. T. Quinn, Kyushu University, for comments on the manuscript. This work was
supported in part by a Grant for Scientific Research
from the Japanese Ministry of Education, Science and
Culture (Project 6377 1448).
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development, penetration, towards, toots, rat, early, amelogenin, germ, immunohistochemical, stage, molar, dental, demonstration, pulp, ameloblasts
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