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Relationship between the quality of fixation and the presence of stippled material in newly formed enamel of the rat incisor.

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THE ANATOMICAL RECORD 208:15-31(1984)
Relationship Between the Quality of Fixation and
the Presence of Stippled Material in Newly
Formed Enamel of the Rat Incisor
A. NANCI AND H. WARSHAWSKY
Department oftlnatomy, McGill University, Montreal, Quebec, Canada
ABSTRACT
Extracellular accumulation of a granular material that is
presumed to be a n organic ‘‘precursor7’to mineralized enamel has been reported. This material, generally referred to as “stippled material,” was observed mainly after immersion fixation with osmium tetroxide. In studies with
perfusion fixation, the presence of stippled material was inconsistent. Therefore, it appeared that the occurrence of stippled material was dependent on the
method of fixation. To test this assumption, tissues were fixed by immersion in
either osmium tetroxide or glutaraldehyde and by perfusion with either glutaraldehyde or a mixture of acrolein, glutaraldehyde, and formaldehyde. It
was found that as the quality of cellular preservation improved, the occurrence
of stippled material decreased. Since no stippled material could be found in
materia1 judged to be well fixed, it was concluded that stippled material is not
a n extracellular precursor to mineralized enamel, but is a breakdown product
resulting from poor fixation.
Enamel is classically described as consisting of a n organic matrix and inorganic hydroxyapatite crystals. The organic matrix is
secreted by the ameloblasts, but there is conflicting opinion about whether it accumulates extracellularly as a n uncalcified
“preenamel.” Fearnhead (1958) first described a granular “precursor substance secreted into the extracellular environment
where fibrillogenesis and mineralization is
taking place.” Watson (1960) was the first to
use the expression “stippled material” to refer to this granular substance. Ronnholm
(1962a,b) suggested that “stippled material
is a precursor of the organic stroma,” which
appeared as thin long septae interconnected
by cross-bridges (Ronnholm, 1962b). The
presence of stippled material has been reported in a variety of species by other investigators (Decker, 1973; Elwood and Bernstein,
1968; Garant and Nalbandian, 1968; Jessen,
1968; Kallenbach, 1971, 1973, 1976; Lester,
1970; Matthiessen and Bulow, 1969; Nylen
et al., 1972; Reith, 1960, 1967; Slavkin et al.,
1976; Travis and Glimcher, 1964). However,
most of these studies used immersion fixation and slow penetrating fixatives such as
osmium tetroxide. With the advent of aldehydes and perfusion fixation, published electron micrographs show that both the
frequency of occurrence and the quantity of
stippled material have decreased.
0 1984 ALAN R.LISS. INC
Since stippled material was visualized
mostly after immersion fixation and since its
presence was inconsistent in perfusion fixation, it seemed possible that the presence or
absence of stippled material could be correlated with the fixation method. The present
study was thus undertaken to investigate the
relationship between the fixation method and
the occurrence of stippled material.
MATERIALS AND METHODS
Male Sherman rats weighing 100 & 5 gm
were used. Teeth were fixed by either immersion or intracardiac perfusion.
For immersion fixation both lower incisors
from a single animal were examined, thus
constituting two observations. In this case,
the alveolar bone overlying the labial surface
of the incisor was partially removed prior to
immersion. Two animals were used for perfusion fixation, and one lower incisor from
each animal was examined.
Received August 26,1982; accepted February 18, 1983.
Address reprint requests to Dr. H. Warshawsky, Department
of Anatomy, McGill University, 3640 University Street, Montreal, Quebec, H3A 2B2, Canada.
Dr. Nanci’s present address is Departement de Stomatologie,
Faculte de Medecine Dentaire, Universite de Montreal, Case
Postale 6209, Succ. A, Montreal, Quebec, H3C 3T9 Canada.
STIPPLED MATERIAL IN ENAMEL
Immersion in Osmium Tetroxide
The rats were decapitated, and the mandibles were dissected a t room temperature.
They were immersed in 2% aqueous osmium
tetroxide’ for 4 hr a t 4°C. Subsequently, the
mandibles were washed in 0.1 M sodium cacodylate buffer containing 0.05% CaClz at pH
7.32, and the lower incisors were dissected
from the surrounding alveolar bone.
Immersion in Glutaraldehyde
After decapitation, the mandibles were dissected a t room temperature and immersed
for 4 hr a t 4°C in 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate containing 0.05%CaCl,, pH 7.3. The mandibles were
washed in the same buffer used for the fixative, and the incisors were dissected from the
alveolar bone. Tissues were postfixed in 2%
aqueous osmium tetroxide for 2 h r at 4°C.
Storage in Lactated Ringer’s Solution Prior
to Fixation by Immersion in Glutaraldehyde
In order to create conditions that might
produce poor fixation, the rats were decapitated and fixation was delayed by storing the
mandibles for 15 min in lactated Ringer’s
solution (Abbott) a t room temperature. After
this treatment, the mandibles (with the labial alveolar bone removed) were fixed for 4
hr by immersion in 2.5% glutaraldehyde
buffered with 0.1 M sodium cacodylate containing 0.05% CaClZ, pH 7.3, a t 4°C. The
mandibles were washed in the same buffer
used for the fixative, and the incisors were
dissected from the alveolar bone. Incisor tissues were postfixed in 2% aqueous osmium
tetroxide for 2 hr at 4°C.
Perfusion With Glutaraldehyde
Rats anesthetized with Nembutal were
prewashed by intracardiac perfusion (by
gravity a t 1 ml/sec) with lactated Ringer’s
Figs. 1-3. Incisor fixed by immersion in osmium tetroxide. Stained with uranyl acetate and lead citrate.
Fig. 1. Mitochondria in the infranuclear compartment are swollen (sm) and some are ruptured (rm).Large
spaces (*) are present between cells. (N, nucleus) x 10,890.
Fig. 2. The intercellular space (*) at the supranuclear
level appears widened and the cell membrane discontinuous (arrows). The Golgi apparatus (GI is well organized
and Golgi saccules are impregnated with osmium. The
rER cisternae are long and interconnected. x 10,890.
Fig. 3. A layer of granular material (gm) is present
around the interdigitating portion of Tomes’ process,
except at the rod (rgs) and interrod growth sites (igs).
Spaces are present among the enamel crystallites.
x 14,740.
17
solution for 30 sec and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate
buffer containing 0.05% CaClZ,pH 7.3, for 10
min at room temperature. After the initial
fixation, the mandibles were dissected and
kept in the same fixative for 3 h r at 4°C. The
mandibles were washed in the same buffer
Perfusion With a Mixture of Acrolein,
Glutaraldehyde, and Formaldehyde
Anesthetized animals were prewashed with
lactated Ringer’s solution for 30 sec and were
perfused with a mixture of 2% acrolein, 2.5%
glutaraldehyde, and 3% formaldehyde (AGF)
in 0.05 M sodium cacodylate buffer containing 0.05% CaC12, pH 7.3, for 10 min at room
temperature. After the initial fixation, the
mandibles were dissected and kept in the
same fixative for 3 h r at 4°C. The mandibles
were washed in 0.1 M sodium cacodylate
buffer containing 0.05% CaC12, pH 7.3, and
the incisors were removed from the alveolar
bone. Incisors were either not postfixed or
postfixed for 2 hr at 4°C in 2% aqueous osmium tetroxide or osmium tetroxide reduced
with potassium ferrocyanide (Karnovsky,
1971).
Following the above fixation procedures,
tissues were dehydrated in graded concentrations of acetone and embedded in Epon 812.
Thin sections (gold interference color) were
cut with a diamond knife on a Reichert Om
U 2 ultramicrotome and stained with 4%
aqueous uranyl acetate (Watson, 1958) for 5
min and with Reynolds’ lead citrate (Reynolds, 1963) for 3 min. Sections were examined in a Philips 400 electron microscope a t
80 kV.
A single segment of the incisor extending
7 mm from the apex of the tooth was cut with
a razor blade and embedded in a long block
mold. The polymerized Epon block was then
cut with a fine jeweler’s saw a t a position
approximately 4 mm from the apex. This produced two blocks, one containing a segment
of the incisor extending from the apex of the
tooth to the region of mid-inner enamel secretion, and another containing the incisor
segment from mid-inner enamel secretion to
almost the end of outer enamel secretion
(Smith and Warshawsky, 1975). In most cases
the region of mid-inner enamel secretion was
selected for study.
’All reagents were purchased from J.B. EM Services Inc. (DorVal, Quebec, Canada).
‘Cacodylate buffer was selected in preference to a phosphate
buffer in order t o avoid any potential interaction with the hydroxyapatite crystallites of enamel. CaClz was added to prevent
the formation of myelinic figures associated with cacodylatebuffered fixatives.
STIPPLED MATERIAL IN ENAMEL
RESULTS
With each fixation method the infranuclear and supranuclear compartments of the
ameloblasts were studied in order to obtain
a n appreciation of the overall quality of fixation. Particular emphasis was placed on evaluating the degree of preservation of Tomes’
processes. The quality of fixation was then
related to the appearance of the enamel and
correlated with the presence, amount, or absence of stippled material.
Immersion in Osmium Tetroxide
At the infranuclear level, the ameloblasts
appeared shrunken with abundant space between cells. Mitochondria were swollen and
some were disrupted (Fig. 1).At the supranuclear level, ameloblasts were separated by
intercellular spaces that varied in width. The
cell membrane seemed discontinuous a t some
points (Fig. 2). The Golgi apparatus appeared
well organized, and saccules were filled with
an electron-dense material. The rER cisternae were long and interconnected (Fig. 2).
Dark and pale-staining granules were distinguishable in the core of Tomes’ processes (Fig.
3). The interdigitating portion of Tomes’ process was separated from the crystallite-containing enamel by a thick layer of material
apparently devoid of crystallites and with a
granular texture. Although this material was
found all around the process, it was usually
not seen at the rod or interrod growth sites
(Fig. 3). Numerous clear spaces were present
among the enamel crystallites (Fig. 3).
Immersion in Glutaraldehyde
At the infranuclear level, the ameloblasts
were separated by a narrow intercellular
space of constant width. Most mitochondria
were intact and their cristae were tightly
packed; however, some were ruptured (Fig.
4).At the supranuclear level, the intercellu-
Figs. 4-6. Incisor fixed by immersion in glutaraldehyde, postfixed with aqueous 0 ~ 0Stained
~ . with uranyl
acetate and lead citrate.
Fig. 4. Mitochondria in the infranuclear compartment are not swollen (m) but some are ruptured (rm).
The intercellular space (arrow) is narrow and of constant
width (N, nucleus). X 10,766.
Fig. 5. At the Golgi level, the intercellular space (arrows) is narrow and of constant width. The Golgi apparatus (G) is well organized. The rER cisternae are long
and interconnected. x 10,766.
Fig. 6. Some granular material (gm) is present at the
periphery of the distal portion of Tomes’ process and at
the interrod growth sites (ips). x 14,573.
19
lar space was also constant and narrow (Fig.
5). The Golgi apparatus appeared well organized (Fig. 5).Much filamentous material was
present in Tomes’ process. Pale and darkstaining granules, although recognizable,
were almost similar in density (Fig. 6). A
thin layer of granular material separated the
interrod prongs from the membrane of
Tomes’ process. The membrane showed shallow bays and numerous coated pits, both of
which were filled with granular material.
With glutaraldehyde immersion, the interrod growth sites contained granular material
(Fig. 6).
Storage in Ringer’s Solution Followed by
Im,mersion in Glutaraldehyde
At the beginning of inner enamel secretion, ameloblasts appeared reasonably well
preserved (Figs. 7-9). The cells were tightly
packed and only a narrow intercellular space
of constant width existed between them (Figs.
7, 8). Most mitochondria were not swollen
and contained tightly packed cristae. Occasionally, focal regions within some mitochondria were vacuolated or ruptured (Fig. 7).
The architecture of the Golgi apparatus
seemed disrupted in some places. Dilated
profiles with a smooth and disrupted membrane were seen (Fig. 8). The interdigitating
portion of Tomes’ process was separated from
the interrod prongs by a very narrow empty
space, and the coated pits along this membrane also appeared empty (Fig. 9). Patches
of granular material were associated with
the most distal part of the process. No granular material was found at the growth sites
(Fig. 9). The organelles within Tomes’ process were not distinct, but dark and palestaining granules could easily be distinguished (Fig. 9).
In a more advanced position of inner
enamel secretion, the ameloblasts appeared
poorly preserved (Figs. 10-12). The intercellular space at the infranuclear level varied
in size. The mitochondria were swollen, some
were ruptured, and some showed disrupted
cristae (Fig. 10). The rER showed short, dilated profiles as well as the more usual Ionger
profiles (Fig. 11). The Golgi apparatus retained its organization. The intercellular
space a t this level was narrow and of constant width (Fig. 11).The interdigitating portion of Tomes’ processes was shrunken and
separated from the enamel (Fig. 12). In some
areas the membrane formed deep, bay-like
invaginations. Many of these invaginations
contained irregular clumps of granular material (Fig. 12).The organelles within Tomes’
process were clearly visible, but it was diffi-
21
STIPPLED MATERIAL IN ENAMEL
cult to distinguish the pale from the darkstaining granules (Fig. 12). The spaces at the
growing end of the interrod prongs contained
small patches of granular material (Fig. 12).
Perfusion With Glutaraldehyde
Both the infranuclear (Fig. 13) and the supranuclear levels (Fig. 14) of the ameloblasts
were tightly packed, and a uniformly wide
intercellular space was present between the
cells. Although most mitochondria were not
swollen and contained tightly packed cristae,
occasional mitochondria were ruptured (Fig.
13). The Golgi apparatus appeared well organized (Fig. 14). A narrow space was often
present between the interrod prongs and
Tomes’ process. The coated pits found on the
membrane of Tomes’ process along these interrod prongs seemed empty (Fig. 15). Small
amounts of dense granular material were
present between rod and interrod enamel,
and occasionally a less-dense granular material was seen a t the rod or interrod growth
sites (Fig. 15). The organelles within Tomes’
process were evident, and pale and darkstaining granules were readily distinguished
(Fig. 15).
Perfusion With Acrolein, Glutaraldehyde,
and Formaldehyde
Ameloblasts were tightly packed both a t
the infranuclear (Fig. 16) and supranuclear
(Fig. 17) levels, and the intercellular space
was of uniform width. The mitochondria1
Figs. 7-9. Incisor was stored in lactated Ringer’s solution prior to fixation by immersion in glutaraldehyde.
Postfixed in aqueous Os04. The micrographs are from
early inner enamel secretion. Stained with uranyl acetate and lead citrate.
Fig. 7. In the infranuclear compartment the mitochondria are not swollen (m?,but some are vacuolated or
ruptured (rm?.The intercellular space (arrows?is narrow
and of constant width. (N, nucleus.) x 10,766.
Fig. 8. At the Golgi level, the intercellular space (arrows) is narrow and uniform in width. Dilated profiles
with smooth and often disrupted membrane (*) are seen
close to the Golgi region (GI. The rER cisternae are long
and interconnected. x 14,573.
Fig. 9. The interdigitating portion of Tomes’ process
is separated from the surrounding enamel by either
empty space, or patches of dense granular material (gm,
white labels) at the most distal part of the process. Less
dense granular material (gm, black label) is present at
the interrod growth sites (igs). x 14,573.
cristae were tightly packed, and no swollen
or ruptured mitochondria were observed (Fig.
16). The Golgi apparatus was surrounded by
long interconnected strands of rER (Fig. 17).
No space was present between Tomes’ process and enamel. The crystallites were directly
in contact with the infolded membrane associated with the rod and interrod growth sites
(Fig. 18).No granular material was observed
between the enamel and the cell membrane.
Perfusion With Aldehyde Mixture and
Postfixation in Potassium Ferrocyanide
Reduced Os04
In order to enhance membrane contrast,
the teeth perfused with mixed aldehydes
were postfixed with potassium ferrocyanide
reduced Os04. This combination produced
images of superior quality. The intercellular
space and the cell membranes were strongly
accentuated, thus clearly defining the limits
between cells at the infranuclear and supranuclear levels (Figs. 19, 20) and between
the cells and the enamel (Figs. 21,221. When
fixation of this quality was obtained, no
spaces were present between the enamel and
Tomes’ processes, and no granular material
was found.
DISCUSSION
Extracellular accumulation of a granular
material related to ameloblasts, and supposedly a precursor of enamel matrix, was described by Fearnhead (1958, 1961) and
Watson (1960). Fearnhead (1961) observed
that as ameloblasts moved away from the
surface of the mineralized dentin the widening extracgllular region became packed with
50- to 70-A granules. This was followed by
the appearance of electron-dense fibers in this
granular material. He proposed that “a granular precursor substance was synthesized
within the cell and then discharged extracellularly where the granules would undergo
fibrillogenesis and then mineralization”
(Fearnhead, 1961). Watson (1960) observed
globular masses of finely stippled material
between dentin and the ameloblasts a t the
beginning of enamel formation. At a more
advanced stage in development, stippled material was localized between the proximal
portions of Tomes’ process of adjacent ameloblasts and between the interdigitating portion and the enamel. Dense ribbon-shaped
profiles, presumably of crystallites, were
embedded in this finely stippled material. He
STIPPLED MATERIAL IN ENAMEL
concluded that “stippled material is recently
synthesized and is not organic matrix leaving the enamel.” However, he pointed out
that stippled material is not always present
in the rat incisor. Ronnholm (196213) related
stippled material to the structured organic
stroma revealed by decalcifying sections of
calcified enamel. He noticed that stippled
material was continuous with thin, long septae that closely resembled the enamel crystals. He thus suggested that stippled material
is a precursor of the organic stroma and that
its stippled appearance “would then correspond to ‘a three-dimensional network formed
by blebs of organic material interconnected
by thin’ bridges.” The concept that stippled
material is a precursor to structured enamel
matrix was further reinforced by Jessen
(1968) and Decker (1973), who observed stippled material adjacent to elliptical tubular
profiles. They proposed that stippled material gives rise to elliptical tubules, the interior of which provides a proper environment
for crystal initiation and growth.
Stippled material was observed in the zone
of presecretion (Reith, 1967; Kallenbach,
1971, 1976; Katchburian and Holt, 1972;
Slavkin et al., 1976);in initial enamel secretion (Watson, 1960; Fearnhead, 1961; Reith,
1967; Kallenbach, 1971, 1976; Katchburian
and Holt, 1972; Slavkin et al., 1976);in inner
enamel secretion (Watson, 1960; Reith, 1967;
Garant and Nalbandian, 1968; Jessen, 1968;
Elwood and Bernstein, 1968; Matthiessen
and Bulow, 1969; Katchburian and Holt,
1972; Kallenbach, 1973,1977); in outer
Figs. 10-12. Incisor was stored in lactated Ringer’s
solution prior to fixation by immersion in glutaraldehyde. Postfixation in aqueous OsO,. The micrographs
are from a more advanced region of inner enamel secretion. Stained with uranyl acetate and lead citrate.
Fig. 10. The mitochondria in the infranuclear compartment are swollen (sm) and vacuolated, and some are
ruptured (rm). The intercellular space (arrows)is of variable width. (N, nucleus.) ~10,766.
Fig. 11. At the supranuclear level, the intercellular
space (arrows) is narrow and of constant width. The
Golgi apparatus (G) maintains its organization. Some
rER cisternae are dilated (*I. x 14,573.
Fig. 12. The interdigitating portion of Tomes’ process
is separated from the enamel by space of variable width
(arrows). Patches of granular material (gm)are found in
this space at places where the process membrane invaginates (igs, interrod growth sites). x 14,573.
23
enamel secretion (Garant and Nalbandian,
1968; Jessen, 1968; Decker, 1973); and in
forming enamel in man (Ronnholm, 1962a,b),
calf (Travis and Glimcher, 19641, and opossum (Lester, 1970). Slavkin et al. (1976) also
found that [3H]-tryptophan, used as a marker
for enamel proteins, is incorporated by ameloblasts, migrates intracellularly, is secreted,
and localizes over a “coarse-textured granular material that was subsequently transformed into the amorphous enamel matrix
material with the associated formation of the
calcium hydroxyapatite crystal nucleation
site.” Stippled material was described between ameloblasts at the level of the proximal portion of Tomes’ process (Watson, 1960;
Reith, 1967; Jessen, 1968; Matthiessen and
Bulow, 1969; Kallenbach, 1973), around the
interdigitating portion of Tomes’ process
(Watson, 1960; Ronnholm, 1962a,b; Reith,
1967; Elwood and Bernstein, 1968; Matthiessen and Bulow, 1969; Decker, 1973; Kallenbach, 1973, 1977), associated with the matrix
between odontoblasts and preameloblasts
(Fearnhead, 1961; Watson, 1960; Kallenbach,
1971, 1976; Slavkin et al., 19761, and intracellularly in membrane-bound granules
(Reith, 1967; Elwood and Bernstein, 1968).In
some cases, material seen between cells a t
the level of the proximal portion of Tomes’
process was called stippled material but was
clearly amorphous in nature (Jessen, 1968;
Kallenbach, 1973).
In most of the above works, tissues were
fixed by immersion in osmium tetroxide
(Fearnhead, 1961; Watson, 1960; Reith, 1960;
Ronnholm, 1962a,b; Travis and Glimcher,
1964; Elwood and Bernstein, 1968; Decker,
1973). Some authors fixed by immersion in
glutaraldehyde or glutaraldehyde-formaldehyde mixture (Garant and Nalbandian, 1968;
Matthiessen and Bulow, 1969; Lester, 1970;
Katchburian and Holt, 1972; Decker, 1973;
Slavkin et al., 1976). Only a few studies used
teeth fixed by whole body perfusion (Jessen,
1968; Kallenbach, 1971, 1973, 1976, 1977).
Hence, stippled material was observed mostly
after immersion fixation either with osmium
tetroxide, which is a slow penetrating agent,
or with aldehydes. In the few cases when
perfusion fixation was used, minimal
amounts of stippled material were observed.
Nylen et al. (1972)pointed out that “stippled
material seems to be seen less frequently the
better the fixation” and that “it must accumulate prior to fixation.” Because of the erratic occurrence of stippled material in the
STIPPLED MATERIAL IN ENAMEL
rat incisor, Kallenbach (1973)suggested that
“it is not a n essential enamel component.”
Besides Kallenbach (1973), who recognized
that “perhaps stippled material is more prevalent in some parts of the secretion stage,”
no effort was made to correlate the presence
of stippled material with the different stages
of enamel secretion. To date, extracellular
material with a granular texture has been
consistently found only in presecretion and
in early initial enamel secretion with all
methods of fixation.
In previous studies stippled material was
identified solely on the basis of its physical
appearance, regardless of location. It was alleged to be a precursor to mineralized enamel purely on generalized morphological appearance. Analysis of published micrographs
leads to the conclusion that stippled material
is not always associated with enamel growth
sites. In fact, Kallenbach (1979)points out that
“it is as often located in presumably nongrowing as in growing sites of enamel.” If
stippled material were a precursor to enamel,
then it would be expected to be found preferentially a t growth sites and not everywhere.
It was observed in the present study that the
presence of stippled material depends upon
the fixative and the fixation method used. Immersion fixation with osmium tetroxide always revealed stippled material; immersion
fixation and perfusion with glutaraldehyde
revealed considerably less stippled material
and perfusion with mixed aldehydes never
produced stippled material. The variable
presence of stippled material observed by others and in this work when perfusion with glutaraldehyde is used is probably related to the
success of the perfusion. If stippled material
Figs. 13-15. Incisor was fixed by perfusion with glutaraldehyde. Postfixation in aqueous0~01.
Stained with
uranyl acetate and lead citrate.
Fig. 13. Mitochondria are not swollen (m); however,
some are vacuolated or ruptured (rm). The intercellular
space (arrow) is narrow and constant in width (N, nucleus). x 10,890.
Fig. 14. At the Golgi level, the intercellular space
(arrow) is narrow and of constant width. The Golgi apparatus (G) seems well organized. The rER cisternae are
long and interconnected. In neighboring cells profiles of
rER are seen to converge toward similar points on the
cell membrane (arrowheads). x 10,890.
Fig. 15. A narrow, but empty space (arrows)separates
the interdigitating portion of Tomes’ process from the
interrod enamel prongs (ir). Small patches of granular
material (gm) are present between rod and interrod
enamel. Granular material of lesser density is present
at the interrod growth site (igs). x 14,740.
25
were a structural entity, then it would be expected to be present with all fixatives and fixation methods, unless there was a preferential
extraction of this material by the fixative. If
extraction did occur, a clear space should be
found in the former location of the material.
Since no spaces were found in the well fixed
tissues (Figs. 16-22), it seemed that preferential extraction was unlikely. From the above
considerations, it may be concluded that stippled material arises as a n artifact of fixation.
Furthermore, it was also found that the presence of stippled material was always associated with poor cellular preservation. Some of
the evidence of poor fixation described in the
present work is found in Watson (1960, Figs. 4,
61, Ronnholm (1962a, Fig. 8) and Reith (1960,
Fig. 8).Poorly preserved Tomes’ processes are
evident in Reith (1967,Figs. 16,17,25),Jessen
(1968, Fig. l), Garant and Nalbandian (1968,
Fig. 171, and Decker (1973, Fig. 1).It is, therefore, suggested that stippled material is seen
in circumstances where preservation is not
ideal and could represent enamel breakdown
prior to completion of fixation.
Despite the absence of stippled material in
inner enamel when mixed aldehydes were
used as a fixative, a substance with a granular texture was observed a t the beginning of
initial enamel secretion (Nanci, 1982). Because a similar granular material has also
been consistently reported in initial enamel
secretion by other authors using different fixative and fixation methods, it is suggested
that this material is not a n artifact. Since this
granular material is not intrinsically electron
dense and requires staining to heighten its
contrast, it must therefore be organic in nature. Indeed, using [3H]-tryptophan, labeled
proteins have been localized over this material (Slavkin et al., 1976). Concomitant with
the appearance of this granular material in
initial enamel secretion, globules of amorphous material were sometimes seen between
adjacent ameloblasts (Warshawsky and Vugman, 1977; Nanci, 1982). Such material was
also seen in inner enamel secretion; however,
it was always localized proximal to the interrod secretion site (Warshawsky and Vugman,
1977;Nanci, 1982).In this position, away from
the secretion site, it is unlikely that it could be
a direct precursor to enamel. The globules of
amorphous material described by Warshawsky apd Vugman (1977)and Nanci (1982)
correspond in position density and texture to
material seen by Reith (1967,Fig. lo), Kallenbach (1971, Fig. 291, Katchburian and Holt
(1972,Fig. 21), and Kallenbach (1973, Figs. 4,
22). The nature of this amorphous material
remains unclear.
Fig. 19. The infranuclear compartment shows close
Figs. 19-22. Incisor fixed by perfusion with a mixture
of acrolein, glutaraldehyde and formaldehyde. Postfixa- apposition between adjacent ameloblasts and between
tion in potassium ferrocyanide reduced 0~01.
The micro- the ameloblast base and the stratum intermedium (SIX
graphs are from a more advanced region of inner enamel Mitochondria (m) show no alterations such as swelling
secretion. Sections stained with uranyl acetate and lead or disruptions. The cytoplasm is not extracted, and the
presence of potassium ferrocyanide within the intercelcitrate.
Mar space clearly delineates the cell boundaries (N,
nucleus). ~10,766.
Figs. 16-18. Incisor fixed by perfusion with a mixture
of acrolein, glutaraldehyde, and formaldehyde. Postfixation in aqueous Os04. The micrographs are from early
inner enamel secretion. Sections stained with uranyl
acetate and lead citrate.
Fig. 16. Mitochondria (m) are not swollen, and none
are ruptured. The intercellular space is very narrow and
uniform in width (N, nucleus). X 10,890.
Fig. 17. The intercellular space at the Golgi level is
very narrow and regular in width. The Golgi apparatus
(G) appears well organized. The rER cisternae are Iong
and interconnected. X 14,740.
Fig. 18. No granular material was present around the
interdigitating portion of Tomes’ processes. The rod and
interrod (ir)crystallites are immediately adjacent to the
cell membrane. x 17,600.
28
A. NANCI AND H. WARSHAWSKY
Fig. 20. The supranuclear compartments of six adjacent ameloblasts show the tubular nature of the Golgi
apparatus (G). The Golgi saccules as well as the intercel-
lular space show the presence of potassium ferrocyi
reduced osmium. x 14,573.
The absence of an extracellular accumulation of a n unmineralized precursor to structured enamel does not preclude the presence
of a precursor form of enamel protein. In fact,
it is generally accepted that nascent ena
proteins are post-translationally modi
through the course of intracellular transF
secretion, extracellular matrix formation,
STIPPLED MATERIAL IN ENAMEL
29
Fig. 21. The use of potassium ferrocyanide clearly
defines the intercellular space between ameloblasts at
the proximal portions of Tomes’ process (pT). The membrane adjacent to interrod growth sites (ips) and rod
growth sites (rgs) is clearly defined. No granular material is present between the cell membrane and rod or
interrod enamel (ir). (iT, interdigitating portions of
Tomes’ processes). x 13,703.
mineralization (see review by Slavkin et al.,
1981).However, from this work it is now clear
that there is no extracellular accumulation of
a preenamel precursor to calcified enamel. In
this regard, enamel differs from the collagenous mineralized tissues that require the presence of a layer of uncalcified matrix prior to
their mineralization.
30
A. NANCI AND H. WARSHAWSKY
Fig. 22. At higher magnification, no granular material is seen at the interrod growth sites (igs) or between
the interdigitating portions of Tomes’ processes (iT) and
the interrod enamel. The organelles in Tomes’ processes,
as well as the relationships between cells in the proxi-
ma1 portion of Tomes’ process ( p a , are judged to be well
preserved. Potassium ferrocyanide clearly defines the
cell membrane tortuousity at the interrod growth site
(igs) (dcw, distal cell web; dG, dark granules; pG, pale
granules). ~23,693.
STIPPLED MATERIAL IN ENAMEL
31
supialis. J. Ultrastruct. Res., 30t64-77.
Matthiessen, M.E., and F.A. Bulow (1969) The ultraThis work was supported by a grant from
structure of human secretory amelohlasts. Z. Zellforsch., 101:232-240.
the Medical Research Council of Canada to
Dr. H. Warshawsky. The authors acknowl- Nanci, A. (1982) A morphological study of Tomes’ process, enamel matrix secretion and the matrix to crysedge the assistance of Miss Anne Bastien,
tallite relationship in the rat incisor. Ph.D. Thesis,
who worked as a summer research student.
McGill University, Montreal.
Nylen, M.U., K-A. Omnell, and C.-G. Lofgren (1972) An
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in developing enamel of the opossum, Didelphis marACKNOWLEDGMENTS
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