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Mitochondrial granule distribution in tooth germ cells.

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Mitochondria1 Granule Distribution in Tooth Germ Cells1
F. S. SAYEGH AND A. ABOUSY
Department of Histology, University of Missouri-Kansas City, School of Dentistry,
Kansas city, Missouri 64108
ABSTRACT
Incisor and molar tooth germs of albino rats sacrificed a t the
eighteenth and twentieth days in utero and one to seven days after birth were
studied with light and electron microscopy. Observations of the various stages of
tooth development in molars established that intramitochondrial granules in
odontoblasts were comparable to the intramitochondrial granules of other hard
tissue cells. These electron-dense deposits appeared in mitochondria in an appreciable number only when odontoblasts become engaged in dentin mineralization. When dentin mineralization was advanced the odontoblast mitochondria
appeared devoid of these deposits. Mesenchymal cells and preodontoblasts of the
pulp were not involved in this activity.
Studies of chondrocytes in rats and mice
(Bonucci, '67; Martin, '68; Matthews, '70) and
chondrocytes in the deer antler (Sayegh et al.,
'73, '74a; Sayegh e t al., '74b) have shown intramitochondrial deposits in these cells. The
nature of these deposits was thought to be
mineral which are associated with calcification. These studies revealed granules in mitochondria within cells a t places where mineralization was taking place.
Because these intramitochondrial deposits
may be valuable indices in determining the
cell potential in sequestrating the inorganic
components of hard tissues, (Bonucci, '67;
Martin, '68; Matthews, '70; Sayegh e t al., '73,
'74a; Sayegh et al., '74b; Talmadge, '70) an attempt was made t o investigate the presence of
mitochondrial deposits in odontoblasts during
dentin mineralization.
MATERIALS AND METHODS
Thirty-eight incisors and molar tooth germs
were obtained from albino rats a t the following time intervals: eighteenth and twentieth
days in utero and one to seven days after birth
(Sayegh and Abousy, ' 7 4 ~ ) .The following
stages of tooth development were selected:
Histo and morpho-differentiation and first increment of dentin formation a t various periods.
Because the incisor is a continuously erupting tooth, our observations were mainly made
on the first molar tooth germs. The eighteenth day in utero represents the histo difANAT. REC., 189: 451-466.
ferentiation of ameloblast in the incisor of
albino rats and the twentieth day in utero represents the initial increment of dentin apposition in incisors and the 1st molar (Sayegh,
'68).
To obtain these stages of tooth formation,
routine histologic procedures (H & E) were
used initially to test the accuracy of the method of tooth germ dissection. Successfully dissected tooth germs designated for EM studies
were placed for two to three hours in a chilled,
phosphate-buffered (pH: 7.4, 0.1 M) 5%glutaraldehyde fixative.
The tissues were rinsed twice in phosphate
buffer and post-fixed for one to two hours with
1.5% phosphate-buffered osmium tetroxide
were then dehydrated with a series of graded
alcohols and embedded in a 60:40 mixture
(A:B) of Epon 812 (Luft, '61).
In three tooth germs, mitochondrial loading
was carried out by fixing the tissue in chilled,
phosphate-buffered 5% glutaraldehyde to
which 5 mM CaCll was added (Matthews et
al., '73).
To avoid possible artifact deposits due to
the phosphate component in the fixative, five
tooth germs were fixed with chilled, S-collidine-buffered (pH: 7,4, 0.1 M) 5% glutaraldehyde. Therefore, these tissues were not exReceived June 8, '76. Accepted Mar. 18, '77.
I This project was partially supported by a grant from General Research fund-Dental School (GRS 3037.2228). Part of this work wan
b a d on an M.S.in oral biology at the University of Missouri.
Dr. Abousy's present address is University of Baghdad-Dental
School, Baghdad, Iraq.
45 1
452
F. S. SAYEGH AND
posed to any external source of calcium or
phosphate during tissue processing. It is possible however, that the aqueous nature of the
fixative could allow for spontaneous precipitation and redistribution of mineral from
tissue fluids.
Thick sections of 1-2 wm were obtained and
stained with Paragon “1301” for orientation.
Ultrathin sections of 40-80 nm were prepared
and stained with an alcoholic solution of
uranyl acetate (Watson, ’ 5 8 ) and followed by
lead citrate (Fahmy, ’68).Grids were examined with a Philips-300 electron microscope
for mitochondrial contents and overall cell ultrastructure.
To determine the nature of the intramitochondrial granules, the microincinceration technique (Thomas and Greenawalt, ’68)
was utilized in tooth germs in which mitochondrial granules were observed. The silicon
monoxide-coated grids used in this procedure
were unstained. It is presumed that if the mitochondrial deposits were organic in nature,
these deposits will dissipate during the ashing
procedure which was carried out for 15
minutes at a 500°C temperature. If the
deposits persisted a t this high temperature
then we could only assume that these mitochondrial deposits are inorganic in nature.
RESULTS
Figure 1 shows an undifferentiated mesenchymal cell observed in the mid-portion of the
dental pulp prior to dentin apposition. Cytoplasmic organelles in general are scanty.
Aside from the very few mitochondria that
are observed in this cell type, other features
consistent with typical mesenchymal cells include: a sparse endoplasmic reticulum, numerous clusters of free ribonucleo-protein
particles and, in general, a lightly stained cell
cytoplasm. Golgi apparatus are distinct in
various cells. The nucleus, an open vesicular
structure, occupies the greater portion of the
cell exhibiting a small amount of heterochromatin accompanied by a large amount
of euchromatin. Prominent nucleoli are frequently observed. The occasionally observed
orthodox and condensed forms of mitochondria are devoid of electron-dense deposits.
It is assumed that mesenchymal or undifferentiated cells are the cells from which the
dentin forming cells, the odontoblasts, will
differentiate. Therefore, we observed these
mesenchymal cells along with those partially
differentiated and finally the highly differen-
A. ABOUSY
tiated odontoblasts for the occurrence of intramitochondrial granules.
Approaching the cell-rich zone at the periphery of the dental papilla, the cells ;appear
to be more differentiated. The fibroblast-like
preodontoblasts appear endowed with the full
complement of the cell organelles and with a
great number of mitochondria. Only a few of
the many preodontoblast mitochondria observed reveal some electron-dense deposits.
Figure 2 depicts five cells from the cell-rich
zone that show an increased number of mitochondria as the most prevalent feature. Figure 3 is a high magnification of cell no. 2 observed in figure 2. There are approximately 20
mitochondria in this section alone, indicating
approximately 2,000 mitochondria per whole
preodontoblast. Examination of the i ndividual mitochondrion again revealed occasional
electron-dense deposits in some mitochiondria
of this cell type. Other cell organelles are
present.
In the odontoblastic zone, where the typical
cell polarity is observed (fig. 41,these tall columnar cells have an ultrastructure similar to
fully differentiated cells. The full complement
of cell organelles is present. A prominent and
well developed granular endoplasmic reticulum is observed on many occasions. Many free
ribonucleoprotein particles are seen in these
odontoblasts but to a lesser degree than in the
undifferentiated mesenchyme cells and preodontoblasts. Golgi vesicles and membraneous
structures lay next to the nucleus. A moderate number of cellular vesicles, glycogen and
lipid droplets are frequently observed in these
cells. Mitochondria are present in both condensed and orthodox forms. However, there
are more mitochondrial aggregations in the
apical portion of the cells. Intramitochondrial
electron-dense deposits are found in these
cells only a t a specific stage. These deposits
are not observed in the odontoblasts, in an appreciable number, until the odontoblasts become definitely involved in dentin formation,
which involves matrix synthesis and mineralization. Distinct intramitochondrial deposits are observed in an appreciable number
only after the initial increment of dentin is
formed and the mineralization front is established. Figures 5 and 6 represent sections of
an odontoblast containing large mitoclhondria
and exhibiting variable numbers of intramitochondrial electron-dense deposits.
These deposits were not seen in the extramitochondrial cytoplasm.
MITOCHONDRIAL GRANULE DISTRIBUTION
At higher magnification (fig. 6 ) these electron-dense deposits appear to be composed of
smaller electron-dense substructures. Cells in
figure 7 are from areas where the numerous
intramitochondrial deposits are needle-like
structures. Bundles of collagen which was
thought to be the collagen fibers which form
the organic portion of the mineralizing predentin are shown a t the bottom of the photograph. Figure 8 shows the same intramitochondrial deposits after microincineration.
Figure 9 shows intramitochondrial granules
before ashing and figure 10 shows the same
granules after ashing. The collagen material
seen in the proximity of the cell membrane is
again thought to be the collagen component of
the predentin which is sectioned in various directions.
DISCUSSION
Basic information regarding the morphological aspects of the cells involved in dentin formation as well as the source of these cells is
necessary to investigate their possible role in
dentin mineralization. Thus, in this report,
the morphological aspect of the mesenchymal
cells of the dental pulp and the predontoblasts
were studied concommitantly with the dentin
forming cells, the odontoblasts.
The source of these odontoblasts has been
partially discussed by Warshawsky and
Smith ('74) and Smith and Warshawsky,
('75). Using 3H-thymidine, the labeled cells
were frequently observed in the undifferentiated and partially differentiated cells of the
dental pulp which are closest to the ameloblasts. It it interesting to note that the odontoblasts themselves did not reveal any mitotic
activity. Also mitochondria of the cell-rich
zone cells which are presumably the partially
differentiated cells, appeared devoid of the
granules. Even the odontoblast mitochondria
prior to dentin formation did not show granules because the odontoblasts were not engaged in the production of the mineral phase
of dentin. We propose that these odontoblasts,
which were devoid of the mitochondria1 granules, were engaged in the production of the
organic phase of the predentin which precedes
the elaboration of the inorganic phase.
In other words, we felt that some odontoblasts perhaps were engaged in the formation
of some increment of unmineralized predentin
first and later on either the same odontoblasts
and perhaps, others in the region will begin
producing the mineral component to form the
mature dentin. These observations were sub-
453
stantiated in both incisor and molar tooth
germs.
The mechanism of dentin synthesis by the
odontoblast using light microscopy (Sayegh,
'67, '68, '69)was studied autoradiographically
with the use of 3H-proline.Ultrastructurally,
with the use of the same collagen precursor,
3H-proline,Weinstock and Leblond ('74) studied the details of the process of dentin formation by the odontoblasts. At two minutes after
3H-proline injection, the collagen precursor
was found to be restricted to rough endoplasmic reticulum. In ten minutes, the labeling of
blood proline declined and the radioactivity
was seen in the spherical part of the Golgi
which later (20 minutes) was seen in the
cylindrical portion. Weinstock and Leblond
('74), found the labeled collagen precursor in
the predentin a t 90 minutes to 4 hours after
the 3H-prolineinjection.
We believe t h a t there is substantial evidence in the literature to claim t h a t t h e odontoblast elaborates the collagenous matrix of
dentin. The real question in this report, however, is whether these odontoblasts have any
role in the formation of the inorganic phase of
the dentinal matrix.
The cell role in the elaboration of calcium
phosphate is a controversial topic. Munhoz
and Leblond, ('74) using 45Ca injected into
rats, claimed t h a t the radioautographic reaction over odontoblasts was due to /3-rays scattering from the heavily labeled matrix. Also
the grain counts were low over the cells and
did not exceed background counts. Therefore
these authors felt that the odontoblast did not
contain detectable labeled bound calcium.
Sayegh et al. ('76) using the scintillation
counting method, demonstrated 45Ca counts
in mitochondria of odontoblasts and papillae
cells ameloblasts and osteoblast of the alveolus surrounding t h e tooth germ. These
counts, representing both free and bound 45Ca,
were obtained a t 5 , 15 and 30 minutes after
45Cainjection in mice.
The role of mitochondria in sequestrating
mineral components, was the object of this
study. Several authors, including ourselves,
have reported on the ability of mitochondria
to form minerals at suitable intracellular and
extracellular concentrations of calcium and
phosphates (Lehninger, '70; Matthews e t al.,
'73; Peachey, '64; Vasington and Murphy, '62;
Greenawalt e t al., '64). These reports were
based on ultra-structural and biochemical
studies of various biological models.
454
F. S. SAYEGH AND A. ABOUSY
It appears then that, in regard to the cell drial deposits were seen in granular form as
role in sequestrating minerals, two different well as in crystalline form. The latter form
points of view do exist in the literature. The was frequently observed in the advanced
present report as well as recent data obtained stages of dentinogenesis. In order to evaluate
from scintillation counting studies (Sayegh e t the possible nature of these deposits we apal., '76) suggest that hard tissue cells do play plied the microincinceration procedures on
some role in the accumulation of calcium both types of mitochondrial deposits. Since
which may be destined t o become the organic the number of granules per mitochondria was
phase of hard tissue matrices. The exact and not changed after ashing, and little or no
complete intracellular pathways have not change was observed in the ashed mitochondrial crystals, we interpreted these deposits to
been worked out.
In the present investigation there was no be mineral in nature (figs. 7-10). We are fully
evidence of mineral accretion in mitochondria aware of the possible effects of the aqueous
of the cells prior to the establishment of the fixatives on the formation of the mitochonmineralization front. This was very decisive drial deposits. Therefore, during the past five
because in the preodontoblast, where the mi- years we used several types of fixatives and
tochondria were the predominant cell organ- these mitochondrial deposits were always
elles, there was no appreciable evidence of present in the odontoblast when dentin minelectron-dense deposits. It was important t o eralization was initiated (Sayegh et al., '76).
note that the number of mitochondria in such These deposits were also reported using a
cells was very large. By comparison with different biological model, the deer antler
other cell types the preodontoblasts revealed (Sayegh e t al., '74a). Whether these deposits
more mitochondria than many hard or soft could be correlated with the crystal containtissue cells. Cell section counting, a method ing vesicle (Sayegh e t al., '76) is a question
previously used by the senior author (Sayegh which requires an answer. Indeed, Anderson
et al., '731, revealed approximately 24 mito- and Sajdera ('76) have recently shown matrix
chondria per section. This indicates that the vesicles containing crystals which give resemnumber of mitochondria per preodontoblast blance to our mitochondrial crystals. Whether
compares very well with the number of mito- or not the mitochondria crystal leaves the cell
chondria of hepatocytes or other cells known is another unanswered question and we are
not ready, as yet, to speculate. We sinlcerely
t o have high metabolic activity.
After the first increment of dentin was feel that research workers in the area of celluformed, definitive intramitochondrial elec- lar mineralization should pay serious attentron-dense deposits were observed in the odon- tion to these questions. Further efforts may
toblasts. These deposits were similar to those prove fruitful in understanding the cell role in
reported by several authors (Bonucci, '67; the process of mineralization.
Martin, '68; Matthews, '70; Sayegh e t al.,
LITERATURE CITED
'73, '74a; Sayegh et al., '74b; Talmage, '70). Anderson, H. C., and 5. W. Sajdera 1976 Calcification of
Higher magnification, however, revealed that
rachitic to study matrix vesicles function. Fed.Proc., 35:
148-153.
these mitochondrial deposits were composed
of some smaller sub-units which may be con- Bonucci, E. 1967 Fine structure of early cartilage calcification. J. Ultrastruct. Res., 20: 33-50.
sidered as primitive protypal, archetypal or Fahmy,
A. 1968 An extemporaneous lead citrate stain
primordial dentinal crystals (Robinson, '75).
for electron microscopy. In: Electron Microscopy Society
of America. Proceedings of the 25th Annual Meeting.
An earlier study (Sayegh e t al., '74b) using
Baton.
high resolution microincineration, showed
J. W., C. S.Rossi and A. L. Lehninger 1964
that these intramitochondrial granules or Greenawalt,
Effect of active accumulation of calcium and phosphate
deposits were mainly mineral.
ions on structure of rat liver mitochondria. J. Cell Biol.,
23: 21-28.
Shapiro and Greenspan ('69) suggest that
these deposits grow within the mitochondria Lehninger, A. L. 1970 Mitochondria and calcium ion
transport. Biochem. J., 119: 129-138.
and leave the cell in some ionic form; how- Luft
, J. H. 1961 Improvements in epoxy resin embedever, the mechanism by which the minerals
ding methods. Biophys. Biochem. Cytol., 9: 409-414.
leave the cell has not been established. The Martin, J.H. 1968 An Electron Microscopic Studly of Intracellular Calcium in the Epiphysis. Ph.D. Dissertation.
mitochondria of hard tissue cells have been
Baylor University, Dallas, Texas.
implicated in the initial phase of the miner- Matthews,
J. L. 1970 Ultrastructure of calcifying tisalization of the skeletal system (Sayegh et al.,
sues. Am. J. Anat., 129: 451-458.
'73, '74a; Sayegh et al., '74b). The mitochon- Matthews, J. L.,J. H. Martin, J. W. Kennedy, 111 and E. J.
MITOCHONDRIAL GRANULE DISTRIBUTION
Collins 1973 An ultrastructural study of calcium and
phosphate deposition and exchange in tissues. In: Hard
Tissue Growth Repair and Remineralization. Elsevier,
Amsterdam, New York, pp. 187-211.
Millonig, G. 1961 Advantages of a phosphate buffer for
O,O, solutions in fixation. Journal of Applied Physics,
v32: 1637.
Munhoz, 0. G.,and C. P. Leblond 1974 Deposition of calcium phosphate into dentin and enamel as shown by radioautography of sections of incisor teeth following injection of W a into rats. Calcif. Tissue Res., 15: 221-235.
Peachey, L. D. 1964 Electron microscopic observations
on the accumulation of divalent cations in intramitochondrial granules. J. Cell Biol., 20: 95-111.
Robinson, B. G. 1975 Personal communication.
Sayegh, F. S. 1967 Healing of the traumatized dental
pulp. J. Dent. Res., 46: 1036-43.
1968 H3-Proline uptake by hard tissue cells.
Program and Abstracts. International Association for
Dental Research Abstract. 46: 185.
1969 3H-Proline uptake by the cells of developing teeth. J. Dent. Res., 48: 257-262.
Sayegh, F. S.,and A. Abousy 1974c Cellular role in hard
tissue mineralization. Anat. Rec., 178: 457.
Sayegh, F. S., R. W. Davis and B. C. Solomon 1974b Mitochondrial role in cellular mineralization. J. Dent. Res.,
53: 581-587.
Sayegh, F. S., K. Porter, G. Sun and G. Sellers 1976 Ca4s
uptake by cdontoblasts mitochondria. J. Dent. Res.
(Special Issue B), 55: B114.
Sayegh, F. S., B. C. Solomon and R. W. Davis 1973 Cellular
455
basis of calcification in the antler tip. J. Dent. Res.,
(Special Issue), 52: 62.
1974a Ultrastructure of intracellular mineral
in the deer's antler. Clin. Orthop., 99: 267-284.
Shapiro, I. M., and J. S.Greenspan 1969 Are mitochondria
directly involved in biological mineralization? Calcif.
Tissue. Res., 3: 100.102.
Smith, C. E.,and H. Warshawsky 1975 Cellular renewal in
the enamel organ and the odontoblast layer of the rat incisor a s followed by radioautography using 3H-thymidine.
Anat. Rec., 183: 523-562.
Talmage, R. V. 1970 Morphological and physiological
considerations in a new concept of calcium transport in
bone. Am. J. Anat., 129: 467-476.
Thomas, R. S.,and J. W. Greenawalt 1968 Microincineration, electron microscopy, and electron differentiation of
calcium phosphate-loaded mitochondria. J. Cell Biol., 39:
55-79.
Vasington, F. D., and J. V. Murphy 1962 Ca" uptake by
rat kidney mitochondria and its dependence on respiration and phosphorylation. J. Biol. Chem., 237: 2670-2677.
Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem.
Cytol., 4: 475-478.
Warshawsky, H., and C. E. Smith 1974 Morphological
Classification of rat incisor ameloblasts. Anat. Rec., 179:
423-446.
Weinstock, M.,and C. P. Leblond 1974 Synthesis, migration and release of precursor collagen by odontoblasts as
visualized by radioautography 3H-proline administration. J. Cell Biol., 60: 92-127.
PLATE 1
EXPLANATION OF FIGURES
1 Electron micrograph of an undifferentiated mesenchymal cell taken from the midportion of the dental papilla. Observe the large nucleus (N), the few mitochondria
(M), an underdeveloped endoplasmic reticulum (E)and many free ribonucleoprotein
particles (R).Original magnification X 15,000.
2 This electron micrograph represents five cells (1-5)from the cell-rich zone. Note the
numerous mitochondria (MIwhich occupy the bulk of the cytoplasmic matrix. Also
note that all mitochondria are devoid of granules. Original magnification x 8,000.
456
MITOCHONDRIAL GRANULE DISTRIBUTION
F. S. Sayegh and A. Abousy
PLATE 1
PLATE 2
EXPLANATION OF FIGURES
3 Preodontoblast from the previous cell zone showing in detail the mitochondria of cell
no. 2. Note the numerous mitochondria (MI and the diminished size of the nucleus (N)
when compared to cell in figure 1. Original magnification X 20,000.
4
458
A composite of a photomicrograph and an electron micrograph of fully differentiated
odontoblasts showing typical cell polarity. Observe the location of the nucleua a t the
distal side of the cell cytoplasm. Full complement of the various organelles are present. Original magnification x 7,500. Insert shows odontoblasts prior to dentin formation. Ameloblast (a), odontoblasts (01, pulp, (p). Original magnification X 400.
MITOCHONDRIAL GRANULE DISTRIBUTION
F. S. Sayegh and A. Abousy
PLATE 2
PLATE 3
EXPLANATION OF FIGURES
5 An electron micrograph showing odontoblasts from the tooth germ at the stage of initial dentin mineralization. Mitochondria are loaded with variable numbers of granules. Original magnification X 12,000.Insert shows dontoblast after the formation
of the first increment of dentin. Odontoblast (o), predentin (p), dentin (d), ameloblasts
(a).
6 Two mitochondria showing several granules. Note that each granule is composed of
small electron-dense deposits thought to be crystal primordia. Original magnification
x 50,000..
460
MITOCHONDRIAL GRANULE DISTRIBUTION
F. S. Sayegh and A. A b u s y
PLATE 3
PLATE 4
EXPLANATION OF FIGURES
7 An electron micrograph showing several mitochondria. Only a few of these mitochon-
dria are labeled and each mitochondria contains several forms of electron dense
deposits. Glutaraldehyde fixed and 0,04 post-fixed. Compare labeled mitochondria
with those in figure 8. Mitochondria (M). Grids were unstained, magnification
X
7,950.
8 An electron micrograph showing the same cell in figure 7 but after microincineration
for 15 minutes. Note that the number of intramitochondrial deposits in these labeled
mitochondria compare favorably with their counterparts in figure 7.Glutaraldehyde
fixed, 0,Od post-fixed. Mitochondria (M). The grids were unstained. Magnification
X 7.920.
462
MITOCHONDRIAL GRANULE DISTRIBUTION
F. S. Sayegh and A. Abouey
PLATE
4
PLATE 5
EXPLANATION OF FIGURES
(MIof
an odontoblast. Each labeled mitochondrion contains several granular forms of electron-dense deposits. Compare the size and shape of these mitochondrial deposits with
those of figure 7. The same electron-dense structures seen here are also seen in
figure 10. Count the number of these mitochondrial deposits with their counterparts
in figure 10.Original magnification x 7,500.
9 An electron micrograph of an unstained section showing several mitochondria
10 An electron micrograph of the same cell seen in figure 9 but after ashing. Compare
the labeled mitochondria in both figures 9 and 10 and note that the same number of
deposits in each mitochondrion (M)is the same. Original magnification X 7,500.
464
MITOCHONDRIAL GRANULE DISTRIBUTION
F. S. Sayegh and A. Abousy
PLATE 5
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