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In vivo experimentation on rat incisor enamel organs through a surgical window.

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THE ANATOMICAL RECORD 210:693-705 (1984)
In Vivo Experimentation on Rat Incisor Enamel Organs
Through a Surgical Window
M.D. McKEE AND H. WARSHAWSKY
Department of Anatomy, McGill Uniuersity, Montreal, Quebec H3A 2B2
Canada
ABSTRACT
Experimental agents administered systemically are costly and
often toxic to animals. An in vivo technique has been developed whereby a
surgical window in the alveolar bone allows selected areas of the rat incisor
enamel organ and underlying enamel to be exposed to various drugs, radiolabeled molecules, and molecular weight markers. Sherman rats weighing 100
gm were anesthetized and the inferior surface of each hemimandible was
surgically exposed. A slow-speed dental hand drill was used to drill a small
hole through the alveolar bone overlying the secretion or maturation zones of
the enamel organ. The wound was closed and during recovery the mechanical
trauma to the underlying tissue moved away from the hole due to the continuous eruption of the tooth. Two to 5 days later the hole was reexposed and
microinjections of 3H-proline, 1251-salmoncalcitonin, vinblastine sulphate, and
normal saline (as control) were administered through the hole with a micromanipulator and a microliter syringe. Radioautographic detection of 3H-proline incorporation in secretory ameloblasts and enamel a t 10 minutes, 30
minutes, 1 hour, 4 hours, 1day, and 2 days after microinjection was identical
to that obtained previously by systemic injection. Two hours after microinjection of vinblastine sulphate the cellular response was again identical to that
following systemic injection; 1251-salmoncalcitonin (M.W. - 3,600D) was used
as a molecular weight marker and was seen to diffuse into the enamel of the
maturation zone at 10 minutes after microinjection. This study has demonstrated the feasibility of this new technique for experimentation on rat incisor
enamel organs.
The incisor of the rat has been shown to be
a n excellent model system in which to study
the complex phenomena associated with
amelogenesis (see reviews in Reith, 1960,
1961, 1963; Watson, 1960; Fearnhead, 1960,
1961a,b; Kallenbach et al., 1963; Warshawsky, 1968, 1971, 1978, 1979; Warshawsky and Smith, 1971, 1974; Weinstock
and Leblond, 1971; Kallenbach, 1973; Skobe,
1976; Warshawsky and Vugman, 1977;
Smith, 1979; Leblond and Warshawsky,
1979). Previously, many methods of in vivo
experimentation on rat incisors have been
devised. Surgical manipulations such as root
resections and transections (Herzberg and
Schour, 1941; Massler and Schour, 1941;
Bryer, 1957; Ness, 1957; Kostlan et al., 1960;
Berkovitz and Thomas, 1969; Berkovitz,
1971) have convincingly implicated the periodental ligament to be directly or indirectly
0 1984 ALAN R.LISS, INC.
associated with the mechanism of eruption
of this tooth. These procedures involved surgically removing a portion of the mandibular
alveolar bone and wounding or removing the
underlying dental tissues. Other ways of
penetrating the alveolar bone have been developed (Melcher, 1970; Gould et al., 1977).
These procedures consisted of drilling a hole
through the alveolar bone to the level of the
periodontal ligament in mouse molars in order to stimulate and locate progenitor cells
of the periodontal ligament.
This study was undertaken in order to determine the feasibility of approaching the
enamel organ of the rat incisor through the
alveolar bone overlying the labial surface of
the tooth. A technique using a slow-speed
Received May 16,1984;accepted July 16,1984
694
M.D. McKEE AND H. WARSHAWSKY
Fig. 1. Enlargement of the right hemimandible of a
Sherman-strain rat killed 24 days after drilling. The
drill used for the surgery is shown above the hole in the
labial alveolar bone (black arrow). The alveolar bone
may be drilled anywhere along its inferior border where
it overlies the secretion or maturation zones of the incisor. The enamel lesion has moved incisally away from
the hole due to the continuous eruption of the tooth and
appears as a shallow pit on the erupted surface of enamel
(white arrow). x 6 . 5 .
dental hand drill equipped with dental burs
has been developed whereby a surgical window in the labial alveolar bone allows selected areas of the rat incisor enamel organ
and underlying enamel to be exposed to microinjections of various drugs, radiolabeled
molecules, and molecular weight markers.
Experimental agents administered systemcally are costly and often toxic to animals.
The main objective of this study was to develop a microinjection technique in which
minute amounts of experimental agents
could be introduced through the surgical
window in the alveolar bone and allowed to
diffuse down and over selected areas of the
enamel organ and underlying enamel. Subsequently, the fate and effects of microinjected experimental agents on the enamel
organ and enamel of the rat incisor could be
compared with the previously described ef-
fects of systemic injections of the same substance. A similar response of the enamel
organ and the enamel to the two different
procedures would establish the feasibility of
the microinjection technique.
MATERIALS AND METHODS
Drilling procedure
Sherman and Sprague Dawley rats weighing approximately 100 gm were used in this
study. The animals were anesthetized with
an intraperitoneal injection of Nembutal and
the inferior surface of each hemimandible
was surgically exposed. Retractors were used
to hold back the masculature and the area
was kept moist with rinses of physiological
saline. A slow-speed dental hand drill
equipped with a straight handpiece and carbide dental burs was used to drill a small
hole (0.75 mm in diameter) through the al-
SURGICAL WINDOW ACCESS TO RAT ENAMEL ORGANS
veolar bone overlying either the secretion or
maturation zones of the enamel organ (Fig.
1).Complete penetration through the alveolar bone into the vascular periodontal space
overlying the enamel organ was determined
by tactile sensation and immediate bleeding
upon breakthrough. The bur was removed
and gauze was placed over the hole for 1
minute to stop the bleeding and the area was
again rinsed with physiological saline. In animals that were killed within 10 minutes
after drilling, the wound was kept moist, but
open. At longer time intervals the wound
was closed, and during recovery the mechanical trauma to the underlying dental tissues
moved away from the hole due to continuous
eruption of the tooth.
Microinjection technique
Animals were drilled in the alveolar bone
overlying the secretion and maturation zones
of the enamel organ. Two to 5 days later the
hole was reexposed to permit microinjections
over the “healthy”ename1 organ and enamel
that had moved underneath the drill site due
to eruption.
A vertical compact micromanipulator
(Brinkman Instruments, Model MM 33) was
mounted on a column stand and a micrometer (Scherr-Tumico, Inc.) was inserted into its
clamping mechanism to allow for extremely
fine vertical movements of a microsyringe
plunger. A 100-plmicrosyringe obtained from
the Hamilton Co. (Cat. No. 710) was fitted
with a 33-gauge needle. A rubber band was
used to keep the plunger of the syringe firmly
applied to the plunger of the micrometer. In
this way, small amounts of the solution to be
injected were drawn into the syringe by the
micrometer. Graduations on the micrometer
were calibrated to the graduations on the
syringe, which allowed dispensing of accurate, reproducible, minute volumes of solution. Due to the small bore size of the 33guage needle several precautions were taken
to insure accurate and consistent microinjections. The needle tip was filed down from its
original bevel to a flat surface using 600grade emory cloth. Both microsyringe and
needle were thoroughly rinsed with physiological saline, rapidly plunged to remove any
compressible air bubbles, and the plunger
was slowly withdrawn so as to partially fill
the syringe with a “buffer zone” of saline.
The syringe was then mounted onto the micromanipulator. Solutions could now be
drawn into the syringe of the micromanipulator assembly. The syringe was filled only
695
immediately prior to injection, and paraffin
film was used to seal the tip of the needle
between injections to prevent evaporation of
the solution from the small-bore diameter at
the tip of the needle.
Microinjection of 3H-proline
L(2,3-3H)-proline(specific activity 32.2 Ci/
mmole) was purchased in 0.01 N HC1 (New
England Nuclear). Under a stream of nitrogen gas, 0.1 ml of solution containing 100
pCi of 3H-proline was evaporated to dryness.
The amino acid was redissolved in 1 pl of
physiological saline to provide for ten microinjections of 0.1 p1 containing 10 pCi of
3H-proline. The solution was slowly drawn
into the microsyringe. Two days prior to microinjection, a minimum of 20 animals were
drilled in the alveolar bone overlying the
secretion zone of the enamel organ in both
hemimandibles and allowed to recover. The
holes were then reexposed and the tip of the
needle was lowered approximately 0.5 mm
into the connective tissue plug that had filled
the lesion in the bone. Approximately 0.1 pl
of solution containing 10 pCi of 3H-proline
was microinjected under a dissecting microscope into the right hemimandible. The
needle was withdrawn immediately, the solution was allowed to “sink in” for 2 mintues, and the wound was closed. The left
hemimandibles were not injected. Animals
were killed at 10 minutes, 30 minutes, 1hour,
4 hours, 1 day, and 2 days after microinjection.
Microinjection of vinblastine sulphate
A group of five rats was drilled in both
hemimandibles as above, two days prior to
microinjection. The right hemimandibles
were microinjected with 0.1 pl of a stock solution of 5 mg/0.3 ml saline of vinblastine
sulphate (Sigma). Injections of physiological
saline into the left hemimandibles served as
controls. All animals were killed 2 hours
after microinjection.
Microinjection of 1251-salrnoncalcitonin
Synthetic salmon calcitonin (approximately 2500 mU/pg) was iodinated with sodium 1251(specific activity 17 pCi/mg; New
England Nuclear), by the Chloramine T
method described by Hunter and Greenwood
(1962). Following these rocedures the super51-salmon calcitonin
natant containing the !
(M.W. - 3,600D) was evaporated to dryness
under a stream of nitrogen gas and redissolved in 2.5% (w/v) bovine serum albumin
SURGICAL WINDOW ACCESS TO RAT ENAMEL ORGANS
in 25 mM TRIS-HC1buffer, pH 7.4. From this
solution, 0.1 pl was microinjected into the
right hemimandibles of two animals drilled
in the alveolar bone overlying the maturation zone 2 days prior to microinjection, and
the animals were killed 10 minutes thereafter.
Tissue processing
All animals used in this study were anesthetized with intraperitoneal injections of
Nembutal and killed by perfusion through
the left ventricle with lactated Ringer’s solution (Abbott) for 30 seconds followed by
perfusion for 10 minutes with a n aldehyde
mixture consisting of 2% acrolein and 2.5%
glutaraldehyde in 0.05 M sodium cacodylate
buffer, pH 7.3. The mandibles were dissected
and immersed in the above fixative for 4
hours a t 4°C followed by washing in 0.1 M
sodium cacodylate buffer containing 0.05%
CaC12, pH 7.3. The mandibles were then decalcified in 4.13% isotonic neutral disodium
EDTA (Warshawsky and Moore, 1967) and
were cut into segments that were extensively
washed in the above 0.1 M sodium cacodylate
buffer. The incisor segments were subsequently postfixed in 2% osmium tetroxide for
4 hours a t 4”C, dehydrated in graded ace-
Fig. 2. A longitudinal section of the right hemimandible showing the original drill site (B) in the alveolar
bone (ab) and the trauma to the underlying tissues (E)
that has moved incisally away from the drill site over 2
days (the large arrow indicates the direction of tooth
eruption). “Healthy” enamel (en) and enamel organ (eo)
have been passively carried underneath the drill site by
the continuous eruption of the tooth. Bone fragments
(small arrows) may be found near the drill site. PS,
periodontal space. x 115.
Fig. 3. Bone lesion (B) drilled over the enamel secretion zone. The animal was killed 2 days after drilling.
“Healthy” enamel (en) and enamel organ (eo) are found
below the bone lesion. Arrows, bone fragments. X89.
Fig. 4. Enamel lesion at 2 days after drilling. The
enamel (en) is completely stripped from the dentin (den),
and cells appearing to belong to the enamel organ continue across the lesion. The enamel thickness is constant
for part of the lesion and may be due to an inhibition of
secretion in this area. Accumulations of red blood cells,
singly stacked or packed in oval clusters not bounded by
endothelium, are found between ameloblasts (black arrows). At the dentinoenamel junction where the enamel
has been removed is a thin layer of dark-staining material (white arrows). c, capillaries; PS, periodontal space.
x356.
697
tone, and embedded in Epon 812. Each segment was orientated for sectioning along the
long axis of the incisor. One-micron-thick sections were cut with glass knives on a Reichert Om U2 ultramicrotome and stained with
toluidine blue or prepared for radioautography (Kopriwa and Leblond, 1962). Thin sections (gold interference color) were cut with
a diamond knife, mounted on copper grids,
and stained with uranyl acetate and lead
citrate. Sections were examined with either
a Siemens Elmiskop 1A or a Siemens 101 a t
80Kev.
The tissues from the ‘251-salmoncalcitonin
experiment were processed as described
above with the exception that the fixitive
contained only 2.5% glutaraldehyde in sodium cacodylate buffer containing 0.05%
CaC12, pH 7.3.
RESULTS
Drilling procedure
Drilling through the alveolar bone of the
rat mandible causes trauma to the underlying dental tissues. The damaged tissues,
however, are passively moved incisally away
from the drill site due to the continuous eruption of the tooth. A gross skeletal preparation
of a drilled right hemimandible and its incisor shows the drill hole in the alveolar bone,
and the trauma to the enamel which is seen
near the incisor tip 24 days after drilling
(Fig. 1).Bone remodeling has occurred a t the
drill site, and the trauma to the underlying
enamel appears as a shallow pit on the
erupted surface of enamel. The distance between the bone lesion and the enamel lesion
(15 mm) corresponds to the normal eruption
rate (651pndday) over a 24-day period (Smith
and Warshawsky, 1975). The bone lesion is
seen in histological sections as a prominent
interruption in the alveolar bone that is filled
with a plug of connective tissue (Figs. 2, 3).
The bones were drilled over the enamel secretion zone and the animals were killed 2
days after drilling. The enamel lesion (Fig. 2)
moved incisally and “healthy” enamel organ
has been passively carried underneath the
drill site by the continuous eruption of the
tooth. The cut edges of bone are clearly visible, and occasional bone fragments may be
observed (Figs. 2, 3). Bleeding often occurs
into the connective tissue of the periodontal
space and extends laterally from the drill
site.
Drilling of the labial alveolar bone in the
rat mandible almost invariably causes le-
698
M.D. McKEE AND H. WARSHAWSKY
sions in the enamel organ and the underlying enamel (McKee, 1984). The lesions
presumably occur when the drill bur penetrates the alveolar bone and plunges a short
distance into the periodontal space. Histologically, the dental tissue trauma appears as
a n altered or ruptured enamel organ overlying a complete discontinuity in the enamel
layer in which the enamel has been completely stripped from the dentin (Figs. 2, 4).
In most cases, the dentin is unbroken. Figure
4 shows a lesion in which the animal was
killed 2 days after drilling. The cells lining
the dentinoenamel junction appear to be derived from the enamel organ as the capillary
network of the papillary layer can be followed over the lesion. The cells resemble
stratum spinosum or stratum intermedium
cells. At the periphery of the lesion the
enamel thickness is constant, indicating a n
inhibition of enamel secretion due to the
trauma. Ameloblasts have decreased in
height but Tomes’ processes appear intact.
Accumulations of red blood cells, singly
stacked or packed in oval clusters not
bounded by endothelium, are found between
ameloblasts. These accumulations conform to
the spatial limitations of the ameloblast
layer.
At the center of the lesion the enamel has
been completely removed. At the dentinoenamel junction and extending only as wide
as the lesion, is a thin layer of dark-staining
material (Fig. 4). This layer completely covers the dentin and varies in thickness. With
the electron microscope (Figs. 5-8), this layer
is bounded by two zones of increased electron
density. In all cases, the arrangement of collagen fibrils a t the dentinoenamel junction is
unusual because the irregular interdigitations of initial enamel with collagen fibrils of
the dentin are not present.
Microinjection technique
Microinjected 3H-proline over the enamel
secretion zone is picked up by the secretory
ameloblasts and incorporated into all proteins being synthesized a t that time, including enamel matrix proteins. At 10 minutes
after microinjection, radioautographic silver
grains are located over the supranuclear region of the ameloblasts (Fig. 9). By 30 minutes, the distribution of the label in the
supranuclear zone is unchanged from above,
but in addition, a weak reaction band is seen
over Tomes’ processes of the ameloblasts (Fig.
lo). At 1 hour after microinjection the reac-
tion band over Tomes’ processes has greatly
intensified (Fig. 11). Silver grains are still
located over the supranuclear zone. By 4
hours, fewer grains are found over the supranuclear zone and the reaction band in the
enamel extends toward the dentinoenamel
junction (Fig. 12). Following the same cohort
of labeled cells as they moved incisally, the
reaction at 1 day is seen over the entire
thickness of the enamel (Fig. 13). Some silver
grains are still observed over the ameloblasts. By 2 days after microinjection, the
cohort is now in outer enamel secretion, and
silver grains are distributed over the entire
enamel layer but very few are over ameloblasts (Fig. 14).
Despite the minute quantity injected into
the drill site, 3H-proline must have entered
the bloodstream because a reaction band is
seen in the predentin at 4 hours in the uninjected contralateral left hemimandible (Fig.
15) from the same animal as the injected
right hemimandible shown in Figure 12.
Ten minutes after microinjection (Fig. 16)
1251-salmoncalcitonin microinjected into the
right hemimandible over the early maturation zone was localized by radioautography.
Most striking is the reaction observed over
the entire thickness of the enamel. Silver
grains are found in greater density over the
outermost enamel but nevertheless extend
completely through the enamel up to the
dentinoenamel junction. The dentin only
shows background labeling. Labeling aIso is
seen in the ameloblasts and in the periodontal space, and to a lesser extent in the papillary layer.
Microinjection of vinblastine sulphate into
the right hemimandible over the enamel secretion zone causes disorganization of organelles within the ameloblasts (Figs. 17-20). In
the supranuclear zone (Fig. 17),marked accumulations of dense-content granules are seen,
rER appears fenestrated or fragmented, and
patches of a granular, dense material are seen
between ameloblasts. These are presumed to
be ectopic secretion sites of enamellike material (Fig. 18). The infranuclear zone contains
mitochondria, rER, and numerous densecontent granules (Fig. 19). Tomes’ processes
also show significant alterations in normal
morphology. The interdigitating portion of
Tomes’ process is completely devoid of organelles (Fig. 20). Although a few membrane infoldings are found, they are not as frequent as
in controls. Microinjection of physiological saline served as controls, and these animals
show normal ameloblast morphology.
SURGICAL WINDOW ACCESS TO RAT ENAMEL ORGANS
Figs. 5-8. Electron micrographs taken a t the center of enamel lesions. The enamel has been
completely removed, and an amorphous layer of material is seen at the dentinoenamel junction
(asterisks).This layer completely covers the dentin (Den), may vary in thickness, and is bounded
by two zones of increased density (arrows, Fig. 8). The irregular interdigitations of collagen
fibrils normally seen at the dentinoenamel junction are not present. Leu, leucocyte. Figures 57, X 10,500; Figure 8, ~52,000.
699
Figs. 9-14. Light microscope radioautographs of secretory ameloblasts (am) at various time intervals after
microinjection of 3H-proline (Pro). At 10 minutes after
microinjection (Fig. 9, x712) grains are present mainly
over the supranuclear cytoplasm. At 30 minutes (Fig.
10, ~ 7 1 2 the
) reaction is seen over Tomes’ processes as
well as the supranuclear zone of the ameloblasts. At 1
hour (Fig. 11, ~ 5 3 4 Tomes’
)
processes and part of the
enamel matrix (en) are heavily labeled, and at 4 hours
) reaction band extends deeper into the
(Fig. 12, ~ 7 1 2the
enamel and the ameloblasts are still labeled. At 1 day
) 2 days (Fig. 14, X534) the reaction is
(Fig. 13, ~ 7 1 2and
seen over the entire enamel matrix but stops at the
dentinoenamel junction. en, enamel matrix.
SURGICAL WINDOW ACCESS TO RAT ENAMEL ORGANS
701
DISCUSSION
A new technique has been developed
whereby the formation of a surgical window
in the labial alveolar bone of the rat hemimandible allows the underlying enamel organ of the rat incisor to be exposed to
manipulation and microinjection of various
experimental agents. The validity of such a n
approach was tested by the application of
several previously published procedures so
that comparisons could be made.
The enamel organ lesion
Early in this study it was noted that complete penetration of the labial alveolar bone
over the zone of enamel secretion by a dental
drill bur invariably produced trauma to the
underlying enamel organ and caused complete removal of enamel underlying the drill
site (Figs. 2-8). Immature enamel from this
zone contains about 30% organic matrix by
weight (see review in Leblond and Warshawsky, 1979) and is referred to as “cheesy
enamel” because of its consistency. The lesion occurs immediately after drilling of the
bone and indicates that the relatively soft
enamel in this zone can be disturbed by
nearby turbulence such a s that caused by the
bur (McKee, 1984). The more rigid matrix of
the dentin remains intact along the entire
length of the lesion. During the process of
repair a layer of amorphous material coated
the surface of the dentin previously covered
by enamel. Part of the mantle dentin seems
to be resorbed along with the enamel, and
the electron-dense lines resemble lamina
limitans seen in regions of functional bone
reversals.
Microinjection of drugs and radiolabeled
precursors
Vinblastine sulphate and 3H-proline were
selected as representatives of a drug and a
radiolabeled molecule, respectively, to assess
the feasibility of the microinjection technique. Microinjection of vinblastine sulphate
caused disruptions in normal ameloblast
morphology and functional activity identical
to those resulting from systemic injection of
vinblastine sulphate (Moe and Mikkelsen,
1977; Takuma et al., 1982; Nanci, 1982) and
colcemid (Karim and Warshawsky, 1979; Nishikawa and Kitamura, 1982).Microinjected
3H-proline was utilized by the secretory ameloblast and incorporated into the organic matrix of enamel in the same way and a t the
Fig. 15. Light microscope radioautograph of odontoblasts and dentin at 4 hours after microinjection of 3Hproline. Silver grains are seen in the predentin (pd) of
the uninjected left hemimandible from the same animal
as the injected contralateral right hemimandible shown
in Figure 12. d, dentin; en, enamel. x510.
Fig. 16. Light microscope radioautograph of the
enamel maturation zone 10 minutes after microinjection
of ‘251-salmon calcitonin. Grains are found over the entire thickness of enamel (en).Labeling also is seen in the
ruffle-ended ameloblasts (r-am) and in the periodontal
space (ps), but to a lesser extent in the papillary layer
(pl). d, dentin. X340.
702
M.D. McKEE A N D €1. W A R S H A W S K Y
SURGICAL WINDOW ACCESS TO RAT ENAMEL ORGANS
same time intervals as systemic injections of
'H-proline (Warshawsky, 1966, 1979; Leblond and Warshawsky, 1979). These results
demonstrate the feasibility of administering
drugs and radiolabeled molecules by the microinjection route.
In some circumstances, the local microinjection technique has advantages over systemic injection. First, experimental agents
that are toxic when administered systemically may be microinjected over selected
areas of the enamel organ and the animal
killed a t any time thereafter. Second, in the
continuously growing incisor of the rat, a
cohort of cells in the enamel organ and the
underlying enamel matrix, a t any specific
developmental stage of amelogenesis, may
be selectively exposed to various experimental agents. Furthermore, due to the incisal
eruption of the tooth, the evolution of this
same cohort and its underlying enamel, and
the fate and effects of the previously administered experimental agents, may be followed
as they pass through the different developmental zones of amelogenesis. Third, intravascular administration of radiolabeled
molecules requires that they be injected in
quantities large enough to reach suitably
high concentrations in all the tissues of the
body. Most systemic studies use dosages of
1,000 pCi, costing about $100 per 100-gm animal. Using the microinjection technique,
only 10 pCi of label, lilOOth of that necessary
for systemic injection, is introduced to a se-
703
Fig. 19. Infranuclear region of secretory ameloblasts
2 hours after microinjection of vinblastine sulphate. This
region contains mitochrondria im), rER, and numerous
dense-content granules (dg). Dense-content granules are
occasionally linked by continuous membrane (arrow).
cw, cell web. x 15,000.
lected volume of cells, thereby eliminating
unneccesary distribution of radioactivity to
all tissues of the body, and eliminating the
risk of radiation damage to sensitive cells
and tissues.
When a single injection of 'H-proline is
administered intravenously, the labeled
amino acid circulates to capillaries overlying
the enamel organ and rapidly appears in the
dental tissues. When the animal is fixed by
perfusion and the tissues processed for radioautography, the free labeled amino acid is
washed out, while the labeled protein is retained. Presumably, a similar route is followed after microinjection. When 3H-proline
is microinjected into the connective tissue
plug that results from healing after the drilling procedure, the labeled proline rapidly diffuses to the ameloblasts of the enamel organ.
Once the amino acid has reached the level of
the connective tissue surrounding the capillary network of the enamel organ, presumably it behaves and is utilized by the enamel
organ in the same manner as amino acids
diffusing from the capillary lumen following
intravenous injection. The only difference is
the continuous high specific activity available after local injection. Concurrent with
the diffusion of 'H-proline into the enamel organ after microinjection, there exists a
similar rapid diffusion of 3H-proline into the
bloodstream as demonstrated by radioautographic reactions over odontoblasts and predentin of the contralateral, uninjected
hemimandible. Because of the appositional
formation of dentin by odontoblasts, it becomes a continuous and stable record of the
availability of labeled amino acids for incorporation into protein (Josephsen and Warshawsky,
1982). Therefore, following
microin'ection of minute quantities ( - 10
pCi) of 3'H-proline into a 100-gm animal, some
of the labeled amino acid rapidly diffuses
into the local vasculature, is removed from
this site by the circulation, and is incorporated by other tissues, as for example the
dentin of the contralateral incisor as a secretory product of the odotoblasts. A similar
amount of radioactivity may have recirculated back to the experimental incisor. However, this small amount of radioactivity did
not obscure the boundaries of the originally
labeled cohort of cells following microinjection.
Fig. 20. Tomes' processes of secretory ameloblasts 2
hours after microinjection of vinblastine sulphate. Tomes'
processes (TP) are completely devoid of organelles, but
some membrane infoldings (arrow) can be found. en,
enamel. x 12,400.
Microinjection of permeability tracers
Several studies have investigated the
permeability of the enamel organ and the
enamel matrix to relatively large macromo-
Fig. 17. Supranuclear region of secretory amcloblasts
2 hours after microinjection of vinblastine sulphate. Accumulations of dense-contentgranules (dg) are seen, rER
appears fenestrated or fragmented, and patches of dense
material (arrow)are seen between ameloblasts. x 12,800.
Fig. 18. Higher magnification of the dense material
seen between ameloblasts similar to that shown in Figure 17. Presumably the location of this material represents ectopic secretion sites of enamel matrix (en). dg,
dense-content granules; cv, coated vesicles; arrows,
coated pits. ~ 5 1 , 7 5 0 .
704
M.D. McKEE AND H. WARSHAWSKY
lecules such as serum albumin (Kinoshita,
1979; Ogura and Kinoshita, 1983; Okamura,
1983). In the enamel maturation zone of the
rabbit both endogenous and exogenous serum
albumin were localized to the ruffled-and
smooth-ended ameloblasts and to the intercellular spaces between them (Okamura,
1983). Serum albumin was not located in the
enamel matrix of the maturation zone although in the secretion zone the surface layer
of enamel was heavily stained by the fluorescent antibody to albumin (Okamura, 1983).
Salmon calcitonin has a molecular weight
of approximately 3,600D and was used in
this study as a molecular weight marker to
investigate the permeability of the enamel
organ and the enamel matrix to proteins of
that size. Radioautography revealed 1251salmon calcitonin over the entire thickness
of the enamel as soon as 10 minutes after
microinjection. The rapidity of appearance in
the enamel suggests that the label is not a
secretory product of the ameloblast and must
represent diffusion either extracellularly or
transcellularly through the enamel organ to
reach the enamel matrix. The results presented here are in agreement with those of
Okamura (1983)in which relatively large exogenous molecules may reach and enter the
ameloblast layer of the enamel maturation
zone. Furthermore, this study presents evidence that the enamel matrix of the maturation zone is completely permeable to a protein
having a molecular weight of approximately
3,600D.
In conclusion, this study has demonstrated
the feasibility of a drilling procedure that
penetrates into the periodontal space overlying the rat incisor enamel organ. In addition,
this procedure shows that surgical penetration of the alveolar bone may cause trauma
to the enamel organ and enamel. This study
has demonstrated the feasibility and advantages of a microinjection technique for in vivo
experimentation on the enamel organ and
enamel of the rat incisor.
ACKNOWLEDGMENTS
The authors wish to thank Dr. P. Bai for
his assistance during this study, and Dr. D.
Goltzman for supplying the iodinated calcitonin. This work was supported by a grant
from the Medical Research Council of Canada t o Dr. H. Warshawsky.
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