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The effect of osmium postfixation and uranyl and lead staining on the ultrastrucure of young enamel in the rat incisor.

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THE ANATOMICAL RECORD 207:l-16 (1983)
The Effect of Osmium Postfixation and Uranyl and
Lead Staining on the Ultrastructure of
Young Enamel in the Rat Incisor
A. NANCI, P. BAI, AND H. WARSHAWSKY
Department ofAnatomy, McGill University, Montreal, Quebec, H3A 2%’
Canada
ABSTRACT
Enamel crystallites are electron opaque without osmium or
heavy metal staining and give a crystalline electron diffraction pattern. Since
the opacity and diffraction pattern are abolished from ultrathin sections of
young enamel by floating on distilled water (Bishop and Warshawsky, 19821,
the possibility that aqueous staining may also remove crystallites was tested.
In addition, the effect of osmium postfixation on crystallite structure was
examined.
Rat incisors fixed by perfusion with a mixture of aldehydes were either
nonosmicated or osmicated prior to dehydration. Incisor segments in the region
of inner enamel secretion were embedded in the same Epon block to ensure
reliable comparison. Osmicated enamel was more intensely stained with toluidine blue and more electron opaque than nonosmicated enamel. No other
structural differences were seen. However, crystallites in osmicated enamel
were more resistant to grid demineralization and electron beam damage.
Routine staining was done by floating sections on solutions of uranyl acetate
and lead citrate; sections were also floated on similar solutions from which the
heavy metals were omitted. These solutions removed the electron opaque
crystallites from the youngest enamel. Stained sections showed electron opaque
crystallite-like structures similar to unstained enamel. When sections that
were extracted by the solutions from which the metals were omitted were
restained, they appeared identical to routinely stained enamel. It was concluded that staining of young enamel removes the crystallites and reveals only
the organic matrix.
Enamel crystallites are intrinsically electron opaque and therefore do not require
staining to be visualized. Yet, few electron
microscope studies of young enamel have
taken advantage of this property (Nylen and
Omnell, 1962; Nylen et al., 1963; Frazier,
1968; Bishop and Warshawsky, 1982). Most
studies have used osmium tetroxide as either
the primary and only fixative (Quigley, 1959;
Reith, 1960; Watson, 1960; Fearnhead, 1961;
Ronnholm, 1962a; Travis and Glimcher,
1964; Glirncher et al., 1965; Boyde, 1967;
Frank and Nalbandian, 1967), or as a secondary fixative after primary fixation with aldehydes (Selvig and Halse, 1972; Bernard,
1972; Decker, 1973; Kallenbach, 1973; Daculsi and Kerebel, 1978; Leblond and Warshawsky, 1979; Warshawsky et al., 1981). In
addition, most of the work used staining with
0 1983 ALAN R. LISS, INC
heavy metals. However, no evaluation has
been made of the contribution by these
agents in producing the final appearance of
the enamel.
The organic matrix of young enamel is
mainly protein and lipid (Stack, 19671, and
like other organic constituents, requires
heavy metal contrasting to be seen. Thus,
stained sections of calcified enamel theoretically should reveal the intrinsic opacity of
the inorganic crystallites together with the
stained organic matrix. Except for Decker
Received September 27, 1982; accepted May 15, 1983.
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.
Address reprint requests to Dr. H. Warshawsky.
2
A. NANCI, P. BAI, AND H. WARSHAWSKY
(19731, none of the works using staining could
describe the presence or distribution of
formed constituents other than crystallites'.
Thus, it can be questioned whether it is indeed possible to demonstrate both components simultaneously in the same preparation.
In this study, the effect of osmium tetroxide
on enamel preservation and structure was
evaluated. In addition, the contributions
made by the commonly used electron microscope stains, aqueous uranyl acetate and lead
citrate, were tested in view of the results of
Bishop and Warshawsky (1982) that distilled
water can remove crystallites from thin sections of enamel. The results indicate that
osmium tetroxide stabilizes the crystallites,
but the solutions commonly used to stain
sections for electron microscopy dissolve
them. Consequently, staining with aqueous
solutions of uranyl and lead is incompatible
with crystallite preservation, and such procedures cannot reveal the relationship between the young inorganic crystallites and
the organic matrix.
tion (Bishop and Warshawsky, 1982). These
were considered to be calcified sections, that
is, they contained electron opaque crystallite-like profiles with no further contrast enhancing procedures.
The effect of postfixation with osmium was
assessed by comparing aldehyde-fixed nonosmicated with aldehydedxed osmicated
specimens. Calcified sections from separate
blocks were examined unstained or stained
in a standardized way with aqueous solutions of 4% uranyl acetate, pH 4.7, for 10
minutes (Watson, 1958) followed by lead citrate, pH 12.5 (Reynolds, 19631, for 5 minutes.
In addition, nonosmicated and osmicated
segments of two separate incisors from the
same rat were embedded together in a common block such that the enamel organs were
in contact. Thus, a single section contained
enamel subjected to both treatments and
comparisons could be reliably made. In this
material, the region of inner enamel secretion was examined.
The effect of staining either nonosmicated
or osmicated enamel was assessed by comparing unstained calcified sections to stained
MATERIALS AND METHODS
calcified sections.
Male Sherman rats weighing approxiTo identify the organic matrix, calcified
mately 100 gm were anesthetized with a n sections were decalcified by floating the gridintraperitoneal injection of nembutal. After mounted sections (Nalbandian and Frank,
perfusion with lactated Ringer's solution for 1962; Ronnholm, 1962a; Travis and Glimabout 1 minute through the left ventricle, cher, 1964) on drops of 1%formic acid in 10%
the animals were perfused for 10 minutes sodium citrate pH 5.0 (Greep et al., 1948), for
with a n aldehyde mixture consisting of 2% 1hour at room temperature.
acrolein, 2.5% glutaraldehyde, and 3% forFinally, the effect of the solutions in which
maldehyde in 0.06 M sodium cacodylate the heavy metals are incorporated was tested
buffer (pH 7.3) containing 0.05% CaC12 or by floating calcified sections for a total of
with 5% glutaraldehyde in the same buffer. 15 minutes on solutions identical in compoThe mandibular incisors were dissected and sition to the stains but containing no heavy
washed in 0.1 M sodium cacodylate buffer metals (solution-extraction). The pH of the
containing 0.05% CaC12, pH 7.3. Some inci- solution for uranyl acetate was 7.6 and for
sors were not osmicated while others were lead citrate 12.9.
postfixed for 2 hours a t room temperature in
All material was examined with a Philips
2% aqueous osmium tetroxide or osmium re- EM 400 operated at 80 kV.
duced with potassium ferrocyanide (KarnovHigh resolution electron probe microanasky, 1971). After dehydration in graded lysis was carried out on conventionally preconcentrations of acetone the teeth were pared specimens, mounted on formvar-coated
embedded in Epon. No deliberate decalcifi- grids. Grids were examined in a modified
cation was performed prior to embedding. JEOL JSM50A scanning electron microscope
Thick sections (1 Fm) were cut with glass capable of scanning-transmission operation
knives and stained with toluidine blue. These and high spatial resolution electron prob? Xwere used to identify the regions of inner and ray microanalysis, in the range of 100 A or
outer enamel secretion that were selected for
analysis. Thin sections (gold interference
'The only exception is the so-called stippled material believed
color) were cut with a diamond knife and by some to be the organic precursor to mineralized enamel.
material is discussed by Nanci and Warshawsky (1983).
quickly removed from the distilled water af- Stippled
These authors conclude that stippled material is an artifact
ter sectioning to prevent crystallite dissolu- caused by breakdown of enamel during fixation.
EFFECT OF HEAVY METAL STAINING ON ENAMEL
less. The instrument incorporates a n 80 mm2
solid-state Si(Li) Princeton Gamma Tech Xray detector (energy resolution 177 eV) located beneath the specimen stage. Vacuum
in the specimen chamber was maintained in
the range of 5 x lop7to 4 x lo-' Torr for Xray microanalysis. The microscope was operated at 25 kV with a fixed beam current of
1 x 10-llA.
Selected area electron diffraction was done
on calcified unstained sections, on calcified
stained sections, on solution-extracted sections, and on solution-extracted stained sections. The selected area aperture was 100 pm
in diameter, covering approximately 5 pm
diameter at the specimen. Diffraction was
done with a camera length of 940 mm at 80
kV in the Philips 400 electron microscope.
3
of separate crystallites (Ronnholm, 1962a) or
central dissolution (Boyde, 1979). The appearance of the enamel crystallites and the
degree of electron opacity in both nonosmicated (Figs. 8, 10) and osmicated (Figs. 9, 11)
teeth were similar. In both cases crystallites
appeared as either sharp dark lines or flat
gray profiles.
Effect of Staining Calcified Sections
The presence or absence of osmium had
only a slight influence on the overall uptake
of heavy metal stains by the young enamel.
The osmicated enamel was somewhat more
dense after staining with uranyl and lead
(Figs. 13, 15) than nonosmicated enamel
(Figs. 12, 14). In these stained, calcified sections crystallite-like structures appeared as
sharp dark lines and flat gray profiles in both
nonosmicated (Figs. 12, 14) and osmicated
RESULTS
(Figs.
13,15) enamel. Staining does, however,
Postfixation With Osmium Tetroxide
impart a granularity to both the sharp dark
The regions of inner and outer enamel se- lines and the flat gray profiles (Figs. 12-15).
cretion were examined. In the case of inner
Grid Decalcification With Formic Acid
enamel secretion, a nonosmicated tooth segWhen grid-mounted sections from regions
ment was embedded together with a n osmicated segment (Fig. 1)to avoid any differ- of inner (Fig. 16) and outer (Fig. 18) enamel
ences that may result from variations in sec- secretion were floated on formic acid all intion thickness and the subsequent handling trinsic electron opacity was removed. Stainof the sections. One-micrometer thick Epon ing the formic acid demineralized sections
sections stained with toluidine blue showed with aqueous uranyl acetate and lead citrate
that osmicated enamel (Figs. 1,3)bound more restored sharp dark lines and flat gray prostain than nonosmicated (Figs. 1, 2). Also, files to the sections (Figs. 17, 19). These crysthere was a marked shrinkage in cell height tallite-like profiles were similar in size, shape
when osmium was omitted (cf. Figs. 2,3).
and distribution to crystallites in calcified
Young enamel crystallites from nonosmi- unstained (Figs. 5, 7, 24, 27, 30, 32) and calcated teeth (Figs. 4,6) were more susceptible cified stained preparations (Figs. 13, 15, 31,
to dissolution in the distilled water of the 33). Since formic acid dissolves enamel crysknife trough than crystals from osmicated tallites, the electron opaque profiles that
teeth (Figs. 5, 7). Nonosmicated crystallites were restored to decalcified sections must
were also more susceptible to electron beam represent binding of the heavy metals to the
damage than osmicated crystallites. Crystal- organic matrix.
lites in the commonly embedded segments of
Effect of Solutions Without Heavy Metals
inner enamel secretion were systematically
(Solution-Extraction)
exposed to beam irradiation for 5 minutes.
The nonosmicated crystallites (Fig. 8) were
Enamel crystallites in calcified sections
extensively sublimated, whereas osmicated from regions of inner (Figs. 24, 30) and outer
crystallites showed few sublimation holes (Figs. 27, 32) enamel secretion appeared as
(Fig. 9). Similar results were obtained on sharp dark lines or pale gray profiles. When
crystallites from separately embedded seg- calcified sections were floated on solutions
ments in the region of outer enamel secretion without heavy metals (Figs. 20, 21, 25, 28)
(Figs. 10, 11). Sublimation created small dis- the sharp dark lines and pale gray profiles
crete holes over the flat surfaces of the rib- were abolished in the region of young enamel
bonlike crystallites. Sublimated crystallites crystallites. The opacity of older crystallites,
viewed on edge gave the illusion of a central located away from Tomes' processes, was only
clear space (Fig. 8). This central clear space sporadically abolished (Figs. 20,21,25). Conwas previously interpreted as either fusion ventional staining of these solution-ex-
4
A. NANCI, P. BAI, AND H. WARSHAWSKY
Fig. 1. A nonosmicated tooth segment (top) is embed- was stained with toluidine blue. Note that osmicated tisded together with an osmicated segment (bottom) in a sues bind more stain than nonosmicated tissues. A, Amecommon Epon block. Both incisor segments arc from the loblasts; D, dentin; E, enamel; 0, odontoblasts. ~ 8 0 .
region of inner enamel secretion. This l-Km thick section
Figs. 2 , 3 . The enamel organs from the nonosmicated
(Fig. 2) and the osmicated (Fig. 3) tooth segments shown
in Figure 1. Equal magnification of comparable regions
shows a marked shrinkage in cell height when osmium
is omitted. The layer of enamel (El is more intensely
stained in the osmicated tooth. A, Ameloblasts. X 800.
EFFECT OF HEAVY METAL STAINING ON ENAMEL
Figs. 4-7. Electron micrographs illustrating the susceptibility of young nonosmicated crystallites to dissolution in distilled water (Figs. 4, 6) and the degree of
protection provided by osmication (Figs. 5,7). The youngest crystallites make up the interrod enamel prongs (ir)
adjacent to Tomes’ processes (T) in the regions of inner
(Figs. 4, 5) and outer (Figs. 6, 7) enamel secretion. Figures 4 and 5 are from commonly embedded incisor segments. The section was floated for a routine length of
5
time on the trough water. Electron opaque crystallites
have been removed from the nonosmicated specimen
leaving clear spaces in the interrod enamel prongs (Fig.
4). No clear spaces are noted in the osmicated segment
in that section (Fig. 5). Instead, opaque crystallites are
seen throughout the enamel. A similar loss of crystallites occurs in nonosmicated outer enamel (Fig. 6) but
not in the osmicated counterpart (Fig. 71. R, Rod.
x 10,023.
6
A. NANCI, P. BAI, AND H. WARSHAWSKY
Figs. 8-11. Detailed view of somewhat older enamel
crystallites (further removed from Tomes’ processes) from
nonosmicated (Figs. 8, 10) and osmicated enamel (Figs.
9, 11). Crystallites in the commonly embedded segments
of inner enamel secretion were systematically exposed
to beam irradiation for 5 minutes. The nonosmicated
crystallites (Fig. 8 ) are extensively sublimated, whereas
the osmicated crystallites show few sublimation holes
(Fig. 9). Similar resistance to beam damage is seen in
the crystallites of outer enamel secretion (compare Fig.
10 to Fig. 11).Sublimination creates small discrete holes
over the flat surfaces of crystallites. Sublimated crystallites viewed on edge give the illusion of a central clear
space (Fig. 8, arrows) previously interpreted as either
fusion of separate crystallites or central dissolution.
~45,562.
EFFECT OF HEAVY METAL STAINING ON ENAMEL
Figs. 12-15. Effect of staining calcified sections with
aqueous solutions of uranyl acetate and lead citrate. The
presence or absence of osmium appears to have only a
slight influence on the overall staining density of the
young enamel. The nonosmicated enamel (Figs. 12, 14)
is paler than the slightly more dense osmicated enamel
(Figs. 13,15). Heavy metal staining does not profoundly
7
alter the unstained images, as sharp dark lines and flat
gray profiles are still seen. Evidence of beam damage
was never seen in these very young “stained crystallite”
images. Figures 12 and 13 are from commonly embedded
segments of inner enamel secretion close to Tomes’ processes (TI.Figures 14 and 15 are from outer enamel
secretion. ~ 4 5 , 5 6 2 .
8
A. NANCI, P. BAI, AND H. WARSHAWSKY
Figs. 16-19. Extraction of calcified sections with
formic acid removes all intrinsic crystallite opacity both
in inner enamel secretion (Fig. 16) and outer enamel
secretion (Fig. 18).The density seen in Figure 18(arrows)
is due to potassium ferrocyanide reduced osmium. Staining these formic acid decalcified sections with aqueous
uranyl acetate and lead citrate restores sharp dark lines
and flat gray profiles (Figs. 17, 19).The granular texture
of these gray images is identical to that seen in stained
calcified sections (Figs. 12-15). Indeed, the images are
virtually indistinguishable. Since the profiles in Figures
17 and 19 cannot be due to hydroxyapatite, they must,
therefore, represent the organic matrix. T, Tomes’ process. ~ 4 5 , 5 6 2 .
EFFECT OF HEAVY METAL STAINING ON ENAMEL
Figs. 20, 21. Effect of the solutions in which uranyl
and lead are incorporated on inner enamel as seen in
the commonly embedded nonosmicated (Fig. 20) and osmicated (Fig. 21) segments. This procedure employed
solutions and times identical to staining with uranyl
and lead, but without these heavy metals. The opacity
due to the young crystallites is removed from the interrod prongs (ir)of both preparations. T, Tomes’ process; R,
rod. x 13.750.
9
Figs. 22,23. When solution-extracted sections are restained with heavy metal-containing solutions (Figs. 22,
23), electron opacity is restored and the images are identical to those seen in formic acid decalcified and stained
sections (see Figs. 17, 19).It must again be concluded
that such opacity cannot be due to hydroxyapatite, but
must represent the binding of heavy metals to the organic matrix of enamel. T, Tomes’ process. ~ 4 6 , 1 2 5 .
10
A. NANCI, P. BAI, AND H. WARSHAWSKY
tracted sections restored electron opaque
images similar in size, shape, orientation,
and distribution (Figs. 22, 23, 26, 29) to the
sharp dark lines and pale gray profiles found
in calcified sections of enamel (Figs. 4, 5 , 6,
7, 24, 27,30, 32). Since the staining solutions
without heavy metals abolished electron
opacity, the dense crystallite-like structures
revealed by the stains must represent heavy
metal binding to the organic matrix. Similarly, the opaque crystallite-like profiles seen
after staining calcified sections (Figs. 12, 13,
14, 15, 31, 33) must also represent prior removal of crystallite opacity followed by binding of heavy metals to the organic matrix.
Electron Probe Microanalysis
High resolution electron probe microanalysis of calcified sections such as those shown
in Figures 5, 24, and 27 revealed a peak for
calcium in the enamel layer (Fig. 34). When
such sections were treated with formic acid,
electron opacity was removed (Figs. 16, 18)
and only a minimal amount of calcium was
detected with the probe (Fig. 34). Therefore,
a loss of electron opacity can be correlated
with a loss of calcium and both parameters
must reflect dissolution of enamel crystallites.
Selected Area Electron Diffraction
Calcified sections of young inner enamel
close to Tomes’ processes (Fig. 35a), similar
to areas shown in Figures 5 and 24, were
analyzed by selected area electron diffraction
(Fig. 35b). The diffraction pattern confirmed
the presence of crystalline material. Adjacent sections stained for a total of 15 minutes
with aqueous uranyl and lead presented similar morphology (Fig. 36a), but selected area
electron diffraction showed that no crystalline material was present (Fig. 36b). Solution-extraction removed the electron opacity
from calcified sections (Fig. 37a), and the absence of crystalline structure was confirmed
by electron diffraction (Fig. 37b). When solution-extracted sections were stained with urany1 and lead, electron opacity was restored
(Fig. 38a), but such material failed to give a
crystalline diffraction pattern (Fig. 38b).
DISCUSSION
It is commonly held that there are two constituents in enamel, the hydroxyapatite crystallite and the organic matrix. While the
crystallites are intrinsically electron opaque
and require no contrasting to be visualized,
the matrix presumably behaves as other organic constituents and needs to be stained in
order to be seen. Therefore, it should be possible to visualize both enamel constituents in
undecalcified, but stained, sections. Previous
attempts to achieve this simultaneous visualization were reviewed by Leblond and Warshawsky (1979). Two major views emerged.
The first postulates that the matrix exists as
a tubule, often visualized as hexagonal in
cross-sectioned profile, with the interior containing the crystallite (Jessen, 1968; Travis
and Glimcher, 1964; Travis, 1968; Decker,
1973). This idea was recently refined and it
is now proposed that a coat of organic matrix
is adsorbed to the outer surface of the enamel
crystallite (Nylen, 1979; Yanagisawa and
Nylen, 1980). The second view postulates
that, a t least in young enamel, the matrix
forms a “crystal ghost” similar to calcified
cartilage (Bonnucci, 1969);that is, it occupies
the same site as the crystallite (Ronnholm,
1962b; Frank, 1979; Nanci and Warshawsky,
1981; Warshawsky and Nanci, 1982).
In order to explore the crystal-matrix relationship it seemed necessary to obtain a better understanding of the role played by the
various contrasting agents normally used to
prepare electron micrographs of the inorganic and organic constituents of enamel.
Visualization of the Inorganic or Crystalline
Constituents
Although the inorganic crystallites can be
seen with no contrast-enhancing treatments,
it has been well documented for bone that
mineral loss occurs from thin sections prepared for transmission electron microscopy
(Boothroyd, 1964; Landis and Glimcher,
1978). Bishop and Warshawsky (1982) have
reported that enamel crystallites are also
susceptible to dissolution even by the water
in the trough of the diamond knife. In addition, they showed that the electron diffraction pattern of young enamel is lost after
staining with aqueous uranyl acetate and
lead citrate.
The present work confirmed the findings of
Bishop and Warshawsky (1982) that young
enamel crystallites are very susceptible to
dissolution in distilled water and to aqueous
staining solutions. Also, this work confirmed
the well-known observation that crystallites
are vulnerable to sublimation under electron
beam irradiation (Boyde, 1979).The only role
attributed to osmication of enamel is that
crystallites are rendered less soluble in thin
Figs. 24-29. Summary of enamel appearances under
various conditions. The region of inner enamel secretion
is at the top (Figs. 24-26, ~ 1 0 , 3 9 5 and
)
outer enamel
secretion is at the bottom (Figs. 27-29, ~ 4 7 , 2 5 0 )Arrows
.
point to electron density due to potassium ferrocyanide.
R, Rod; ir, interrod prongs; T, Tomes’ process. Figs. 24,
27. In these calcified unstained sections, enamel crystallites are intrinsically electron opaque and appear as thin
flat ribbons or as sharp needle-like densities. Figs. 25,
28. Solution-extractionresults in an abolition of electron
opacity due to the loss of young enamel crystallites. In
Figure 25 older crystallites (*) are only partially removed. Figs. 26, 29. When solution-extracted sections
are conventionally restained, electron opaque images
similar in size, orientation and distribution to those in
calcified sections are now present. Since solution-extraction dissolves enamel crystallites and abolishes electron
opacity these opaque crystallite-like structures must
represent organic matrix.
12
A. NANCI, P. BAI, AND H. WARSHAWSKY
Figs. 30-33. Comparison between osmicated enamel
in a calcified unstained section (Figs. 30,32) and stained
section (Figs. 31, 33) in the region of inner enamel secretion (Figs. 30, 31) and outer enamel secretion (Figs. 32,
33) very close to Tomes’ processes. Staining does not
significantly alter the images seen in unstained preparations. Since staining solutions dissolve young enamel
crystallites, the images in Figures 31 and 33 must represent the site of organic matrix. Since the pattern of
the organic matrix is identical to the unstained crystallites, it is concluded that the matrix occupies the same
site as the hydroxyapatite crystallites. T, Tomes’ process;
R, rod; ir, interrod enamel. x 100,237.
EFFECT OF HEAVY METAL STAINING ON ENAMEL
280
13
..
200 -.
160 -.
La--+---?-
-i
---f--40 E m 100 120 140 160 180 200 220 240 260
CHRNNEL NO,
I
ZEl
I
Fig. 34. Energy dispersive X-ray spectra of young inner enamel, examined at a screen magnification of x
6,000 over areas on the sections of 20 X 25 pm (raster
mode) and 300-second integrated detecting time periods.
The recorded X-ray intensities (abscissa)were calibrated
at 20 eV per channel. The upper trace was generated
from osmicated, unstained sections not exposed to formic
acid. The lower trace was obtained from similar sections
decalcified on formic acid. The principal Cak,, peak was
not detected in the formic acid decalcified enamel (arrow). The Cak, peak (normally occurring at - channel
201) was too small to be detected in either of these
spectra. The X-ray peak positions of osmium and phosphorus (if present) overlap in the region of channel 100.
Detectable sulfur is intrinsic to the tissue; chlorine derives from the support film over the grid. Other details
of the microanalysis method have been published (Landis and Glimcher, 1982).
sections and osmium reduces their vulnerability to electron beam damage. No alteration was noted in the actual appearance of
the enamel with or without osmication. Although the ability of the enamel to bind toluidine blue and heavy metals was increased
after osmication, uranyl and lead staining
showed no major difference in crystallite appearance. Except for a granularity, no additional material was revealed in the stained
sections when compared to the unstained sections, a n observation similar to that reported
by Ronnholm (1962b).
electron opacity formerly attributed to crystallites. High resolution electron probe microanalysis showed a loss of calcium from the
enamel layer, thus confirming that formic
acid had decalcified the thin section. Uranyl
acetate and lead citrate staining of the formic
acid-decalcified thin sections should reveal
the organic constituent. Indeed, staining restored electron opaque images to the sections, but these were indistinguishable from
the original crystallites.
Since distilled water and formic acid can
decalcify thin sections of enamel, the aqueous
staining solutions themselves might also be
capable of decalcifying enamel sections.
Thus, utilizing staining solutions of the exact
composition, but without uranyl and lead,
also removed the electron dense crystallite
images from thin sections and abolished the
crystalline electron diffraction pattern. Urany1 and lead restaining of these extracted
sections produced images indistinguishable
from formic acid decalcified and stained enamel. From these experiments it was concluded
Visualization of the Organic Matrix
Constituents
Staining of calcified sections of enamel did
not reveal a n organic constituent that differed from the crystallites seen without
staining. Therefore, in order to visualize the
organic matrix alone, sections were decalcified with formic acid. Floating grid-mounted
thin sections of calcified enamel for 1 hour
on formic acid resulted in a complete loss of
14
A. NANCI, P. BAI, AND H. WARSHAWSKY
Fig. 35. a) Area of inner enamel close to Tomes’ processes (T) selected for electron diffraction from a calcified, unstained section. x 11,700. b) Diffraction pattern
from the selected area shown in (a). The concentric lines
or maxima confirm the presence of crystalline material.
The discontinuity in the concentric lines demonstrates a
preferred crystallite orientation.
Fig. 36. a) Area of inner enamel close to Tomes’ processes (T) from a calcified section stained with aqueous
uranyl acetate and lead citrate for a total of 15 minutes.
No major structural differences are seen when compared
to Figure 35a. x 11,700. b) Diffraction pattern from the
area shown in (a). The absence of lines or maxima indicates that no crystalline material is present.
that routine staining cannot be used to re- Leblond and Warshawsky (1979). Yanagiveal the relationship between the crystallite sawa and Nylen (1980) favor the view that
and the matrix, because these staining solu- “crystals in developing enamel bind organic
tions decalcify the section leaving only the material to their surfaces, a n d . . . it is this
stained organic matrix. It is thus not possible material which is visualized a s structural
to visualize both constituents of young en- elements in decalcified sections of enamel.”
The present work could not demonstrate a
amel simultaneously on the same section.
structured organic matrix that was separable from the crystallite images. No stained
Crystallite-Matrix Relationship
material was present between crystallite imThe question of how the hydroxyapatite ages and no coat of material was detected a t
crystallite relates to the organic matrix has their surface. Thus, only a “ghost” of organic
been recently discussed by Nylen (1979) and material within the crystallite image could
EFFECT OF HEAVY METAL STAINING ON ENAMEL
Fig. 37. a) Area of inner enamel close to Tomes’ processes (T)from a calcified section that was solutionextracted for 15 minutes. Note the absence of needle-like
densities. x 11,700. b) The diffuse haloes in this diffraction pattern confirm the absence of crystalline material
in the selected area shown in (a).
15
Fig. 38. a) Selected area of inner enamel close t o
Tomes’ processes (T) from a calcified section that was
solution-extracted and stained for 15 minutes with urany1 and lead. The needle-like densities have been restored. X 11,700. b) The diffuse diffraction pattern shows
that the needle-like densities in (a) are not crystalline.
be demonstrated. On theoretical grounds, an organic matrix septa. This organic material
organic stroma within a ckystallite is dis- would not interfere with the visualization of
puted because of space restrictions within crystal lattice fringes by high resolution electhe lattice of the crystal structure (Nylen and tron microscopy.
Omnell, 1962). However, the crystalline
structure could grow unimpeded on the surACKNOWLEDGMENTS
face of an organic template. Supposing that
such a template is a thin flat strip of matrix
This work was supported by grants from
capable of initiating or promoting crystallite the Medical Research Council of Canada. The
growth, then crystalline hydroxyapatite authors are deeply indebted to Dr. William
could grow on all surfaces. Thus, as previ- J. Landis, Harvard Medical School, for his
ously proposed by Ronnholm (1962b), the collaboration with the electron probe and for
young crystal would grow on both sides of a n providing Figure 34.
16
A. NANCI, P. BAI, AND H. WARSHAWSKY
LITERATURE CITED
Bernard, G.W. (1972) Ultrastructural observations of initial calcification in dentine and enamel. J. Ultrastruct. Res., 41:l-17.
Bishop, M.A., and H. Warshawsky (1982)Electron microscopic studies on the potential loss of crystallites from
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young, effect, osmium, enamel, uranyl, ultrastrucure, rat, staining, postfixation, leads, incisors
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