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Radioautographic demonstration of receptors for epidermal growth factor in various cells of the oral cavity.

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THE ANATOMICAL RECORD 222:191-200 (1988)
Radioautographic Demonstration of Receptors for
Epidermal Growth Factor in Various Cells of the
Oral Cavity
MOON-IL CHO, YU LIN LEE, AND PHILIAS R. GARANT
Department of Oral Biology and Pathology, School of Dental Medicine, State University of
New York at Stony Brook, New York 11794-8700
ABSTRACT
Mouse iodinated epidermal growth factor (EGF)was localizedby light
and electron microscopic radioautography in basal cells of oral epithelium, papillary
cells of the enamel organ, periodontal ligament fibroblasts, preodontoblast precursor
cells, and preosteoblasts of the alveolar bone of 13-day-old Sprague-Dawleyrats. The
specificity of binding in these cells was suggested by an observed reduction of about
90% in the labeling when excess unlabeled EGF was injected along with the 125I-EGF.
In contrast, fully differentiated cells, such as ameloblasts. odontoblasts, and osteoblasts, were only poorly labeled. Quantitative analysis of the light microscopic radioautographs revealed that the papillary cells had the highest level of labeling (5.5grains
per 100 pm2 of cell area). The significance of the rather high labeling of the preosteoblasts of the alveolar bone and the fibroblasts of the periodontal ligament is unknown. However, the well-known effect of EGF in producing precocious eruption of
teeth may be a consequence of an effect on these two cell types.
Epidermal growth factor (EGF) was originally isolated from male mouse submandibular glands (Cohen,
1962). It is a single-chain polypeptide of 53 amino acids
with a molecular weight of 6,045 Da (Carpenter and
Cohen, 1979). EGF is a potent mitogen for various cell
types, both in vitro and in vivo (Carpenter and Cohen,
1979; Das, 1982). It is well documented that EGF binds
to specific receptors on the cell membrane of its target
cells and is internalized into an endosomal compartment (Carpenter and Cohen, 1976; Haigler, et al., 1979).
One of the earliest findings with respect to the in vivo
effects of EGF was the precocious eruption of incisors
when it was administered to newborn mice (Cohen, 1962).
EGF administered in vivo binds to cells in the enamel
organ of developing teeth (Martineau-Doize,et al., 1987).
The in vitro binding of EGF to mouse and human odontogenic tissue a t various stages of development has also
been studied (Thesleff, 1987; Thesleff, et al., 1987; Partaneu and Thesleff, 1987). These recent demonstrations
of the binding of EGF to various odontogenic tissues
suggests that this growth factor may have a significant
role in the differentiation of the enamel organ and dental follicle mesenchyme. The recent observation of the
binding of EGF in the apical tissues of a developing
human premolar root (Thesleff, et al., 1987) is in good
agreement with the observed association of precocious
tooth eruption and EGF (Cohen, 1962; Frindik, et al.,
1985). However, the mechanism responsible for precocious eruption remains unknown.
In this study, we have identified the cell types that
bind EGF in vivo during the development of teeth. With
lZ5I-EGFradioautography, a high number of binding
sites for EGF were detected on the cell membranes of
basal cells of oral epithelium, papillary cells of the
enamel organ, periodontal ligament fibroblasts, and
@ 1988 ALAN R. LISS, INC
preosteoblasts. On the basis of these observations, we
speculate that accelerated eruption of teeth resulting
from treatment with EGF may be due t o a direct and/
or indirect effect on periodontal ligament fibroblasts
and preosteoblasts.
MATERIALS AND METHODS
lodination of Epidermal Growth Factor
Mouse EGF (receptor grade) was purchased from Collaborative Research, Inc. (Waltham, MA) and radioiodinated by the chloramine T technique (Hunter and
Greenwood, 1962; Carpenter and Cohen, 1976)to a specific activity of approximately 100 pci/pg immediately
before use.
Injection of l2’I-EGF
A total of three 13-day-old Sprague-Dawley rats
weighing 23.5 +- 0.1 g were used. lZ5I-EGFwas injected
through a jugular vein under ether anesthesia. In order
to study the in vivo localization of specific binding sites
for EGF by radioautography, two rats were injected
with 100 pci of lZ5I-EGF(S.A. = approximately 100 pci/
p.g) in 0.1 ml of 0.05 M potassium phosphate buffer, pH
7.5, containing 0.075 M NaCl. To assess the specificity
of binding, a control rat was injected with 0.1 ml of 0.05
M potassium phosphate buffer, pH 7.5, containing 100
pci of KI-EGF and an excess (50 pg) of unlabeled EGF,
and 0.075 M NaC1.
Tissue Preparation for Radioautography
At 5 min after administration of the iodinated growth
factor, the animals were anesthetized by intraperitoReceived November 18, 1987; accepted March 2,1988.
192
M.-I. CHO, Y.L. LEE, AND P.R. GARANT
TABLE 1. Quantitative analysis of light microscopic radioautographic silver
grains over various cell types of the developing first maxillary molars of
13-day-oldrats
Experimental group
Control group
# Grains Mean # # Cells # Grains Mean #
counted counted per cell counted counted per cell
# Cells
Cell types
Oral epithelium basal cells
Enamel organ:
Presecretory ameloblasts
Stratum intermedium
Postsecretory ameloblasts
Papillary cells
pulp:
Odontoblasts
Bone:
Osteoblasts
Preosteoblasts
Periodontal ligament fibroblasts
117
1,734
14.8
103
401
3.9
90
63
88
95
48
31
196
4,505
0.5
0.5
2.2
47.4
91
409
4.5
111
135
1.2
89
80
116
63
864
1,496
0.7
10.8
12.9
73
134
131
228
1.8
1.7
neal injection of Nembutal (1 mg/l00 g body weight)
prior to an intracardiac perfusion with lactated Ringer's
solution for 20 sec. This procedure was immediately
followed by perfusion with 2.5% glutaraldehyde in 0.1
M sodium cacodylate buffer, pH 7.4, for 10 min. The
maxillae were dissected free of the surrounding tissues
and further fxed in Karnovsky's fixative (Karnovsky,
1965) for an additional 3 hr. After rinsing in 0.1 M
cacodylate buffer for 10 min, the maxillae were decalcified for: 4 days at 4°C in 0.1 M EDTA containing 3%
glutaraldehyde. The areas containing the first maxillary molars were then mesiodistally sectioned into 1mm-thick slices. These slices, along with tissue containing the developing third molars, was postfixed for
1%hours in 1%OsO, in s-collidine buffer, pH 7.4. Tissues were then dehydrated in a graduated series of cold
ethanols and propylene oxide prior to infiltration with
Poly Bed mixture a t room temperature for 4 hr and
were placed in flat embedding molds containing fresh
embedding mixture. Polymerization was accomplished
at 60°C for 48 hr.
Electron Microscopic Radioautography
To prepare electron microscopic radioautographs, the
loop method of Caro and Van Tubergen (1962) was employed as described previously (Cho and Garant, (1981b).
Thin sections approximately 100 nm thick were cut from
four blocks of each animal and were coated with a crystalline monolayer of Ilford L4 emulsion. After exposure
at 4"C, the sections were developed with Microdol-X.
QuantitativeAnalysis of Radioautographs
Identification of cell types with specific binding sites for
epidermal growth factor
A total of 12 blocks (two blocks from each maxillar;
i.e., eight blocks from the experimental rats and four
blocks from the control rat) were selected and prepared
for light microscopic radioautographs under conditions
identical to those described previously. Photographs were
taken at x 1,000 in a Zeiss photomicroscope (Carl Zeiss,
Inc., NY)and printed at a final magnification of x 5,000.
The number of cells and silver grains over these cells
were counted on the photographic prints. The cell types
Tissue Processing for Morphology
subjected to quantitative analysis of radioautographs
Three 13-day-oldrats weighing 23.0 f 0.1 g were an- are listed in Table 1.
esthetized by intraperitoneal injection of Nembutal (1 Binding sites for epidermal growth factor per 100 pm2 of
mgI100 g body weight) before intracardiac perfusion cytoplasmic surface area
with 2.5% glutaraldehyde in 0.1 M sodium cacodylate
In an attempt to study the relative number of binding
buffer, pH 7.4, for 10 min. All subsequent procedures
were identical to those described in tissue preparation sites for EGF present on different cell types, only those
for radioautography. However, for morphological study, cell types with high mean values of silver grains were
en bloc staining in 1%uranyl acetate in 0.1 M maleate subjected to quantitative analysis. The areas were
buffer, pH 6.2, was added between postfixation in 1% measured on the photographic prints with a Kontron
OsO, and dehydration in alcohol (Cho and Garant, MOP analyzer after cell boundaries were drawn with
1981a). One-micrometer sections cut in a mesiodistal a red color marker. The mean value of silver grains per
100 pm2 of cell was obtained for each cell type (Table 2).
plane were stained with 1%toluidine blue.
tight Microscopic Radioautography
Three t o four 1-km thick sections from each block
were mounted on glass slides, coated with Kodak NTB2 liquid emulsion, exposed at 4"C, and developed as
previously described (Cho and Garant, 1981b). The radioautographs were subsequently stained with 1%toluidine blue in sodium acetate buffer.
RESULTS
Light Microscopic Morphology
In 13-day-oldrats used in this study, the mesial cusp
of the first maxillary molar was located directly under
the oral epithelium. Root development was well advanced. The collagenous fibers of the periodontal ligament were arranged obliquely from the root surface
EPIDERMAL GROWTH FACTOR RECEPTORS IN THE ORAL CAVITY
193
TABLE 2. Quantitative analysis of light microscopic
radioautomaphic silver grains over unit area (100 pn2)
of cell volume
Cell types
# Grains/100 fim2
Basal cells of oral epithelium
Papillary cells in association with postsecretory
ameloblasts
Preosteoblasts
Periodontal ligament fibroblasts
3.4
5.5
1.9
2.1
Light microscopic radioautographs from the experimental animals contained numerous silver grains, mostly
restricted to the basal cells (Fig. 2a). Within the basal
cells, the majority of silver grains appeared to be located
at both the basal and lateral portions of the cells (Fig.
2c). The apical portion of the cell was relatively free of
silver grains (Fig. 2c).
Electron microscopic radioautographs confirmed these
observations (Fig. 2d). The number of silver grains on
the cells of the basal layer was reduced dramatically
from 14.8/cell in the experimental tissues t o 3.9kell in
the control tissues (Fig. 2b, Table 1).
Enamel organ
Light microscopic radioautography of the enamel organ in the experimental animals clearly showed that
neither the presecretory, secretory, and postsecretory
ameloblasts nor the cells of the stratum intermedium
were labeled (Fig. 3a, b). Heavy labeling was observed
along the peripheral region of all papillary cells (Fig.
3b). Ultrastructural morphology of this region showed
a large number of microvillilike structures projecting
into the intercellular spaces. Electron microscopic radioautography revealed specific localization of silver
grains along the cell membrane proper, but not on the
microvilli (Fig. 3d, el.
The number of silver grains was greatly reduced over
papillary cells of the control rat, although the pattern
of labeling remained the same as in the experimental
group (Fig. 3c, Table 1).
Bone
Fig. 1. Light micrograph showing the mesial developing root of the
first maxillary molar of a 13-day-old rat x 100. Low-magnification micrograph (inset)illustrates the mesial half of the developing tooth with
its enamel organ under the oral epithelium (ep). x 23. The rectangular
area of inset shows an area similar to that shown in Fig. 1. A = ameloblasts; AB = alveolar bone; D = dentin; E = enamel; ES = enamel
space; HR = Hertwig's root sheath Od = odontoblasts; P = pulp; PD
= predentin; PDL = periodontal ligament.
In areas of active bone formation, light microscopic
radioautography from both the experimental and control animals demonstrated that clearly identifiable osteoblasts (including osteoclasts; result not shown)
remained unlabeled (Fig. 4a, b). However, prominent
labeling was observed over fibroblastic cells near the
osteoblasts (Fig. 4a). A small number of grains remained in association with these cells in the control
group (Fig. 4b, Table 1).
Electron microscopic radioautography clearly confirmed that the labeled fibroblastic cells were relatively
immature cells, perhaps preosteoblasts in the process
of migrating toward the prebone surface between adjacent osteoblasts (Fig. 4c).
coronally toward the alveolar bone. Odontoblasts were Periodontal ligament
The developing periodontal ligament of the first maxlocated along the predentin surface in the pulp (Fig. 1).
illary molars of 13-day-old rats was characterized by
Radioautographic Localization of Binding Sites for '251-EGF
fewer collagen fibrils and more fibroblasts, compared
Oral epithelium
with a fully developed and functional periodontal ligThe oral epithelium covering the first developing ament (Fig. 5a). Periodontal ligament fibroblasts exmaxillary molars was a typical stratified squamous epi- hibiting a high degree of polarization and orientation
thelium (Figs. 2a,c).
were heavily labeled along their cell boundaries in the
194
M.-I. CHO, Y.L. LEE, AND P.R. GARANT
Fig. 2. Ramoautographs showing localization of '25I-EGF on oral epithelium. a: Light microscopic radioautograph of oral epithelium from an
experimental animal reveals a heavy labeling with silver grains on the
basal cells (arrowheads).x 120. b The number of grains is dramatically decreased on the basal cells from the control animal x 120. c: Cells
of the cornified (C),granulosum (G),and spinosum (S)layers are free
of labeling, in contrast to heavy labeling of basal layer (B).X 900. d
Eledon microscopic radioautography demonstrates localization of silver
grains (arrowheads) along the basal and lateral protions of the cell
membrane of the basal cells (B) from an experimental animal. x 2,700.
S = spinosum layer; F = fibroblasts.
Fig. 3. Radioautographs showing the localization of ‘“I-EGF on the
enamel organ. a: Light microscopic radioautograph of developing third
molar shows that both presecretory amelohlasts (A) and cells of the
stratum intermedium (SI) of an experimental animal remain unlabeled.
x 350.D = dentin. b Papillary cells (P)of an experimental animal are,
however, heavily labeled, whereas only a small number of grains are
present on the papillary cells from the control animal ( c ) x 400. A =
amelohlasts; B = blood vessel. d Electron microscopic radioautograph
of papillary cells (P) from an experimental animal localizes the silver
grains on the cell membrane. x 1,200.A = ameloblasts; M = microvilli.
e: The silver grains (arrowheads) are present on the cell membrane
of papillary cells, hut not on the microvilli (MI.X 2,700.
196
M.-I. CHO, Y.L. LEE, AND P.R. GARANT
Fig. 4. Radiographs showing localization of lZ5I-EGFon bone tissue.
a: Preosteoblasts (arrowheads)from an experimental animal demonstrate heavy labeling. x400. The number of grains on those from the
control animal (b) are reduced dramatically. x 400. Osteoblasts (Ob)
and osteocytes (OC) remain unlabeled. B = bone; pb = prebone. c:
Electron micrsocopic radioautography of an experimental animal exhibiting a preosteoblast (POB) migrating (arrow) toward prebone (PB)
between two osteoblasts (OBI. x 2,000.
Fig. 5. Radioautographs showing the localization of 1251-EGFin the
periodontal ligament. a: Light microscopic radioautographs from an experimental animal demonstrating numerous silver grains over periodontal ligament, x 120. AB = alveolar bone; D = dentin. b: Only a
small number of grains are present on the periodontal ligament from
the control animal. X 120. AB = alveolar bone; D = dentin. c: Most
silver grains are located over cell membrane of periodontal ligament
fibroblasts (F). x 500. d Electron microscopic radioautograph illustrating the localization of silver grains (arrowheads)near the cell membrane of the fibroblasts (F). X 2,000.
198
M.-I. CHO, Y.L. LEE, AND P.R. GARANT
experimental animals (Fig. 5a, c). A marked reduction
in number of silver grains was observed over these cells
from the control group (Fig. 5b, Table 1).Electron microscopic radioautography clearly revealed the localization of silver grains along the cell membrane of
periodontal ligament fibroblasts (Fig. 5d).
Odontoblasts
Odontoblasts at different stages of cytodifferentiation
were identified along the forming root surface (Fig. 1).
The precursor cells, aligned next to the root sheath, did
not show any special orientation or evidence of alignment to the epithelial cells (Fig. 6a). As differentiation
progresses, odontoblasts become closely packed, elongated, and polarized and show evidence of secretion of
the dentinal matrix (Fig. 6b). Mature odontoblasts,
characterized by the presence of odontoblastic processes, form predentin and dentin (Fig. 6c).
Light microscopic radioautography of 1251-EGFdemonstrated that neither newly differentiated odontoblasts nor fully mature odontoblasts bind EGF (Fig. 6b,
c). However, a small number of grains were found over
the cells in the subodontoblastic layer, as well as over
odontoblast precursor cells close t o the root sheath (Fig.
6a, c).
Quantitative Analysis of Radioautographs
Silver grain counts in experimental tissue indicated
that a small number of grains (less than 2.2 grains per
cell) were observed over ameloblasts, stratum intermedium cells, osteoblasts, osteocytes, osteoclasts (result not shown), and odontoblasts (Table 1).However,
a high number of grains per cell was observed over
papillary cells (47.4) in association with the postsecretory ameloblast, basal cell (14.8) of oral epithelium,
periodontal ligament fibroblast (12.9), and preosteoblast (10.8) (Table 1).
In contrast, the number of silver grains localized over
these cells types was signficantly reduced in control
tissue to approximately one-tenth the number recorded
in the experimental groups (Table 1). This difference
may indicate that the silver grains observed in the experimental animals represent specific binding sites for
EGF.
The relative number of specific binding sites for EGF
present on each cell type was obtained by counting the
silver grains per unit area (100 pm2)of cell volume for
each cell type. It was found that papillary cells showed
the highest number (5.5),followed by basal cells of oral
epithelium (3.4), periodontal ligament fibroblasts (2.1),
and preosteoblasts (1.9) (Table 2).
DISCUSSION
It has been established that 1251-labeledhormones
and EGF maintain biologic activity and an ability to
bind to their specific receptors on the cell membrane
Fig. 6. Radioautographs depicting localization of '"1-EGF durign den(Carpenter and cohen, 1976; Bergeron et al., 1977, 1978; tinogenesis.
a: The epithelial root sheath (E)and the subadjacent odonBarazzone et al., 1980; Warshawsky et al., 1980; Fehl- toblast precursor cells (arrow) slightly labeled. x 400. P = pulp cells.
mann et al., 1982; Silver et al., 1982; Nanney et al., b Newly differentiated odontoblasts (Od) are unlabeled, while some
1986). Radioautography has been used for the in vivo silver grains are loeated on subodontoblasticfibroblasts, shown at higher
in 6e (arrow). x 400. c: Mature odontoblasts (Od) show
and in vitro demonstration of receptor sites for calci- magnification
no labeling. A small number of silver grains are associated with fibroblast
tonin (Warshawsky et al., 1980), insulin (Bergeron et (arrow) adjacent to the odontoblast layer. ~ 5 0 0D. = dentin; OP =
al., 1977), growth hormone (Barazzone et al., 1980),and odontoblastic process; PD = predentin.
EPIDERMAL GROWTH FACTOR RECEPTORS IN THE ORAL CAVITY
199
EGF (Nanney et al., 1984, 1986; Martineau-Doize et EGF on root formation and vascular changes during
al., 1987; Thesleff, 1987; Thesleff et al., 1987; Partinen tooth eruption. In addition to root formation and vasand Thesleff, 1287).Binding sites for lZsI-EGFin normal cular changes, the development of the periodontal lighuman skin were located primarily on the mitotically ament and the formation and resorption of bone have
active basal keratinocytes and appeared in diminished been regarded as major factors responsible for tooth
numbers as the degree of differentiation of these cells eruption (Ten Cate, 1980a, b; Avery, 1987).Among these
progressed (Nanney et al., 1984,1986). Our results sup- factors, the periodontal ligament has been regarded as
port these findings; in addition, our electron micro- the most likely source for generating the force required
scopic radioautographic results demonstrate that EGF for tooth eruption. Although the generation of traction
receptors are located primarily along the basal and lat- within the periodontal ligament remains unclear (Ten
eral cell membranes of the basal cells and only rarely Cate, 1980a, b), it has been suggested that collagen
molecules and/or the fibroblasts themselves may be the
on the apical surface of these cells.
Recently, McKee et al. (1986) investigated the entry major sources of contractile force generation (Thomas,
and penetration of various I251-labeledproteins with dif- 1964; Bellows et al., 1982a, b; Ten Cate, 1980a, b).
Considering that collagen fibrils are a product of fiferent molecular weights into the enamel organ and
enamel of the rat incisor. They reported heavy labeling broblasts and that their organization and turnover are
of papillary cells by lZ5I-EGFbut were unable to deter- manipulated by fibroblasts, it may be concluded that
mine whether this labeling was specific or nonspecific. EGF may influence the rate of tooth eruption by its
By comparing the labeling in animals injected with lZ5I- effect on periodontal ligament fibroblasts. FurtherEGF only and animals injected with both W-EGF and more, a mitogenic effect on preosteoblasts might inan excess of unlabeled epithelial growth factor, Mar- crease the rate of alveolar bone formation, which could
tineau-Doize et al. (1987) obtained evidence suggesting also contribute to tooth eruption.
a ligand-receptor interaction. Our results agree with
their findings.
ACKNOWLEDGMENTS
Of interest is the fact that of all cells examined in
This research was supported by grants #DE03745
our study, the papillary cells had the highest number and #DE06165 from the National Institute of Dental
of binding sites for epithelial growth factor (5.5 per 100 Research, National Institutes of Health. We thank Mrs.
pm2 unit area of cell volume), even exceeding that of Champa Codipilly for her excellent technical assisthe basal cells of the oral epithelium (3.4 per 200 pm2)>.tance. The help of Mrs. Kris Vandenberg in preparation
The physiologic signficance of this abundance of EGF of the manuscript is greatly appreciated.
binding by papillary cells remains to be established.
Note added in proof: The possible roles of EGF on
Although the mitogenic effect of EGF, its binding and tooth eruption was further studied by Rodes et al. (Deintracellular degradation have been intensively studied vel. Biol. 121:247-252,1987) and Topham et al. (Devel.
in cultured fibroblasts (Carpenter and Cohen, 1974, Biol. 124532443, 1987).
1979), the in vivo localization of specific binding sites
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In view of the small amount of EGF that is bound by
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125-138.
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epidermal, factors, growth, cavity, demonstration, radioautography, receptors, oral, cells, various
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