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Fine structure of an end organ in the periodontal ligament of the mouse incisor.

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Fine Structure of an End Organ in the Periodontal Ligament
of the Mouse Incisor
Histological Laboratory, Faculty of Medicine, University ofdmsterdam, l e Const.
Huygensstraat 20, Amsterdam and Department of Periodontology, Faculty
of Dentistry, University ofdmsterdam, Louwesweg 1,
Amsterdam, The Netherlands
Nerve endings were observed between collagen fiber bundles in
the alveolar compartment of the periodontal ligament. In close relation to these
endings, cells were observed with a rounded cell body from which cytoplasmic extensions protruded, each forming part of a sheath around the terminal region of
a small nerve fiber. The nuclei of these cells occupied an eccentric position in the
cytoplasm and were characterized by a typical kidney shape. In the cell's center
a prominent Golgi complex was present. In the peripheral cytoplasm ladder-like
structures with a periodicity of 160 nm were occasionally observed. The cell
bodies were surrounded by a basal lamina, while the cytoplasmic extensions
investing the nerve endings were surrounded by a n envelope consisting of alternating electron dense and electron translucent layers containing material that
stained with PAS and Alcian Blue. Where these envelopes bordered on the connective tissue, areas of filamentous material with a periodicity of 100-150 nm
were occasionally found. The end organs may be considered to form part of a receptor system.
From a physiological study of the rabbit
mandibular incisor, Ness ('54) concluded that
three types of mechanoreceptors may be distinguished in the dental tissues, each characterized by a specific response after applying a
certain stimulus to the tooth. So far, little
information has become available in the literature as to the localization and nature of receptors in the periodontal ligament of the continuously erupting incisor. Hattyasy ('59)observed various types of nerve fibers in the
ligament of the rat incisor, but he was unable
to discern any special cellular organization in
relation to these structures. In a previous
study of the rat incisor we described cells in
the periodontal ligament which, on the basis
of certain ultrastructural characteristics,
were considered to have a sensory function
(Beertsen et al., '74). In subsequent studies of
the lingual part of the periodontal ligament of
the rodent incisor we observed similar cells in
the mouse. I t was considered of interest to
study these cells in more detail in order to get
some insight into their overall ultrastructure,
their spatial relationship to other components
of the ligament and, if possible, to get some
idea as to their functional significance.
ANAT. REC., 189: 73.90.
Four male CBA mice, five months old, were
used. The animals were anaesthetized by
tracheal intubation with a mixture of NzO,
Halothane (Hoechst),and Ozand received 125
E heparin and 0.1 ml of a solution of 0.5%
NaNOz intravenously to prevent vascular
contraction. They were perfused through the
left cardiac ventricle with a solution containing 5.56% glutaraldehyde and 0.89% OsO, in
28 mM cacodylate buffer (pH 7.0) for 15
minutes a t a rate of 2.5 ml per minute. The
lower jaws were removed and demineralized
at 4OC for 18 days using 0.1 M EDTA in
cacodylate buffer containing 2.5% glutaraldehyde (pH 6.0).After subsequent washing in
cacodylate buffer, appropriate portions of the
incisor with the surrounding periodontal tissues were dissected out and postfixed with 1%
OsOl in cacodylate buffer for 60 minutes. The
specimens were dehydrated through an ethanol series and embedded in Epon 812. Sagittal
and transverse sections (oriented as previously described: Beertsen et al., '75) were cut
with a diamond knife on a LKB ultrotome
Received Jan. 7, "77. Accepted Feb. 16, '77.
and, after staining with uranyl acetate and
lead citrate, examined in a Philips EM 300.
For orientation, 0.5- to 1-pm sections were
used, stained with methylene blue.
For general inspection of the ligament sections from mandibles, fixed in 10% neutral
buffered formalin, demineralized in 12.5%
EDTA and embedded in Paraplast were used
(for description of this material see Beertsen, '75).
The lingual part of the periodontal ligament of the incisor consists of two compartments, one related to the tooth and the other
to the alveolar bone (fig. 1). Neural elements
were observed exclusively in the alveolar compartment of the periodontal ligament, i.e., in
the connective tissue areas around and between the vascular spaces (figs. 2, 3). In the
avascular tooth related part of the ligament
(which moves along as the incisor erupts,
Beertsen, '75) neural elements were not
found. Although nerve endings, characterized
by dense accumulations of mitochondria, were
seen along the whole length of the incisor,
they were more frequent in the proximal region of the ligament up to the first molar than
in the distal region. Cells associated with the
terminal region of nerve fibers had a roundish
cell body with a diameter of 7-10 pm. Their
nuclei showed deep indentations giving them
a kidney shape and had an eccentric position
in the cells (figs. 2-51.Because of the typical
shape of their nucleus we propose that these
cells be called K(idney1-cells. From the cell's
cytoplasm, extensions protruded each showing a close spatial relationship to the terminal
region of an unmyelinated nerve fiber (figs. 4,
5). Where we could follow the course of the
nerves it seemed that the terminal region of
the axons originated from myelinated nerve
fibers (fig. 3). K-cells associated with nerve
terminals contained a prominent granular
endoplasmic reticulum and showed a pronounced Golgi apparatus (fig. 6). While the
Golgi apparatus was generally situated in the
central part of the cell in close proximity to
the nucleus, the endoplasmic reticulum had a
less fixed position, extending from the cell
body into the peripheral parts of the cytoplasm. In the center of the K-cell a pair of centriolar profiles was present and occasionally
one of these organelles was seen to be associated with a cilium (fig. 7).Invariably a number of microtubules was observed, many of
which radiated from the centriolar region into
the cytoplasmic processes.
Microfilaments 5-7 nm in diameter were
usually found in the peripheral parts of the
cytoplasm (fig. 81, particularly in the cytoplasmic processes. Occasionally ladder-like
structures composed of filamentous material
were observed having a cross-banded appearance with a periodicity of 160 nm, each period
marked by a n electron dense band (figs. 9 , l O ) .
These structures were located directly beneath the plasma membrane and resembled
leptomeric organelles described for the rat incisor (Beertsen et al., '74). Subdivision of the
periods as described in our earlier report could
not be clearly discerned. Along the cell membrane of both cell body and cytoplasmic processes, many vesicles were observed. These
were rather uniform in size, with a diameter
of about 60 nm. Vesicles were seen on both
aspects of the cellular processes, i.e., in relation to the cell-connective tissue interface as
well as the cell-nerve fiber interface (fig. 14).
Occasionally coated vesicles were seen.
As mentioned above the K-cells possessed
cytoplasmic extensions, each forming part of
a thin sheath surrounding a nerve terminal.
The extensions thus arranged had a thickness
of a t least 150 nm. The cytoplasm of these
sheaths mainly contained vesicles, a fine
array of microfilaments, some microtubules,
ribosomes and occasionally a mitochondrion.
The cytoplasmic extensions enveloping the
nerve terminal did not always form a continuous sheath. Small gaps occurred through
which parts of the axoplasm, rich in microfilaments and vesicles, were seen to bulge outward (fig. 11).On the other hand adjacent
cytoplasmic sheaths were sometimes found to
contact each other by means of junctions
which had the appearance of zonulae occludentes (fig. 12).
The terminal region of each axon contained
neurofilaments and neurotubules. The most
striking feature, however, of the endings was
the presence of dense accumulations of mitochondria. These organelles varied greatly in
size and internal structure. Beside the usual
slender crista-type mitochondria, we found
giant size mitochondria containing typical arrays of membrane folds (fig. 13).Some of these
folds seemed to have a tubular shape (fig. 14).
In some mitochondria elongated crystal-like
structures of some unknown material were
present (fig. 15).
The cell bodies of the K-cells were surround-
ed by basal lamina-like material (fig. 5). In
certain places, particularly where the cell extensions formed a sheath around the nerve
endings, irregular layers of lamellar, fluffy
material were seen (figs. 4, 14, 16) forming a n
envelope measuring up to 2 p m in diameter.
The thickness of the individual electron dense
lamellae varied from 30-60 nm. They appeared to be separated from each other by
electron translucent areas. In paraffin sections these envelopes could be discerned with
the light microscope as they contained material staining with Alcian blue (pH 3.5) and
PAS (periodic acid Schiff). Where envelopes or
basal lamina bordered on the collagen fibers,
islands of typically arranged filaments were
sometimes seen. These filaments had a diameter of about 8-9nm and were orientated more
or less parallel to each other. They formed
crossbanded aggregates with a periodicity of
100-150 nm (figs. 17,18).
The end organs described in this study are
similar to those reported for the periodontal
ligament of the rat incisor (Beertsen et al.,
'74). The nerve endings contain dense accumulations of mitochondria which may indicate that they are sensory receptors (Munger,
'65). The cells associated with these terminals
contain many vesicles along the plasma membrane and are surrounded by basal laminalike material. In some of these cells leptomeric organelles with a periodicity of 160
nm were observed as has been found previously in the rat. The cells can easily be recognized
on the basis of their rounded cell body and the
typical kidney shape of the nucleus. The presence of many vesicles along the plasma membrane may indicate pinocytosis. Another possibility could be that these vesicles are related
to a process of secretion, e.g., of material constituting the envelopes which surround the
cellular processes. In this respect it is of
interest that the cells possess an extensive
Golgi apparatus. Many vesicles, however,
were also found along the cell membrane facing the nerve ending. In a study of encapsulated nerve endings Patrizi and Munger ('65)
suggest that such vesicles may be instrumental in the movement of ions around nerve
fibers. The envelopes staining with Alcian
blue and PAS are of unknown functional significance. One may speculate that these structures somehow modify pressure exerted on the
end organ not unlike the function of the la-
mellae in the Pacinian corpuscle. More specifically one could envisage some degree of distribution over the transducer membrane, increasing the number of excited active sites
(Loewenstein, '60).
Fibrillar cross-banded structures with a periodicity of 100-150nm as found on the boundary between the envelopes and the surrounding connective tissue, have been described in
various organs, both under normal and pathological conditions (for review see Banfield et
al., '73). In a study of Descemet's membrane of
the cornea Jakus ('56) considered this material to be collagen. Cauna and Ross ('60) described similar structures in Meissner's corpuscle and suggest that this fibrillar material
"may act to maintain a tautness within the
whole structure, which could enhance the responsiveness of the corpuscle to physical deformation." On the other hand it may well be
that the occurrence of this material is merely
incidental, a possible consequence of interaction of collagen with components of the
ground substance, since it is well-known that
interaction of collagen with various substances may result in unusual types of aggregation (Gross et al., '52).
One may wonder whether the end organ described in the present study is identical to
neural elements already reported in the literature. Although several types of nerve endings seem to occur in periodontal ligament,
most of the descriptions have been based on
light microscopical observations of nerve
fibers only. The various types of endings may
be tentatively classified into three morphologically distinct groups:
1. Convoluted endings: ovoid structures
having a length of ca 20-40 p m and a width of
ca 20 pm, which are composed of twisted or
coiled nerve fibers (e.g., Berkelbach van der
Sprenkel, '35; Lewinsky and Stewart,'37;
Rapp et al., '57; Falin, '58; Kizior et al., '68;
Griffin, '72).
2. Endings of fibers, terminating as lamellar, knob-, spindle- or leave-like structures
(e.g., Lewinsky and Stewart, '37; Bernick, '57;
Hattyasy, '59).
3. Fine delicate fibers which seem to terminate as free endings (e.g., Lewinsky and
Stewart, '37; Bernick, '57; Rapp et al., '57;
Hattyasy, '59; Kizior et al., '68).
The nerve endings observed in the present
study (figs. 2, 3) resemble group 2 endings
with thickened, rounded enlargements as described by Lewinsky and Stewart ('37) for the
rabbit and the lamellar endings as reported by
Hattyasy ('59) for the rat. Although we did
not quantify the number of K-cells, we have
the impression that this cell type, which is associated with nerve endings, is very common
in the alveolar compartment of rodent incisors. We confirm the finding of Hattyasy
('59) that nerve endings terminating as lamellar or knob-like enlargements are numerous. The dense accumulations of mitochondria
may indicate that these endings are sensory
in nature. It is not known, however, what significance, if any, has to be given t o the
unusual shapes and sizes of mitochondria in
the terminal axoplasm. Mitochondria containing crystal-like structures have been described in a variety of tissues (Tandler and
Hoppel, '72). Preliminary observations in our
laboratory indicate that a similar end organ
occurs in the periodontal ligament of the
mouse molar. Therefore this type of end organ
does not seem to be typical for the rodent incisor. Although the end organs may be considered to form part of a receptor system, in
our opinion it is impossible to characterize the
nerve endings and associated cells in a more
detailed functional sense on the basis of morphologic studies only.
We would like to thank Doctor J. James for
his helpful comments on the manuscript and
Mr. C. E. Gravemeyer for careful handling of
the photographic material.
Banfield, W. G., C. K. Lee and C. W. Lee 1973 Myocardial
collagen of the fibrous long spacing type. Arch. Pathol.,
95: 262-266.
Beertsen, W. 1975 Migration of fibroblasts in the periodontal ligament of the mouse incisor as revealed by autoradiography. Arch. Oral Biol., 20: 669-666.
Beertsen, W., V. Everts and A. van den Hooff 1974
Fine structure and possible function of cells containing
leptomeric organelles in the periodontal ligament of the
rat incisor. Arch. Oral Biol., 19: 1099-1100.
Beertsen, W.,
V. Everts and J. M.Houtkooper 1975 Frequency of occurrence and position of cilia in fibroblasts of
the periodontal ligament of the mouse incisor. Cell Tissue
Res., 163: 415-431.
Berkelbach van der Sprenkel, H. 1935 Zur Neurologie des
Zahnes. 2.Mikr. Anat. Forsch., 38: 1-86.
Bernick, S. 1957 Innervation of teeth and periodontium
after enzymatic removal of collagenous elements. Oral
Surg., 10: 323-332.
Cauna, N., and L. L. Ross 1960 The fine structure of Meissner's touch corpuscles of human fingers. J. Biophys.
Biochem. Cytol., 8:467-482.
Falin, L.I. 1958 The morphology of receptors of the tooth.
Acta Anat., 35: 257-276.
Griffin, C. J. 1972 The fine structure of end-rings in human
periodontal ligament. Arch. Oral Biol., 17: 785-797.
Gross, J.,F. 0. Schmitt and J. H. Highberger 1952 In vitro
fibrogenesis of collagen. In: Fourth Conference on Metabolic Interrelations, New York. E. C. Reifenstein, Jr., ed.
Josiah Macy Jr. Foundation, New York, pp. 32-57.
Hattyasy, D. 1959 Zur Frage der Innervation der Zahnwurzelhaut. Z. Mikr. Anat. Forsch., 65: 413-433.
Jakus, M. A. 1956 Studies on the cornea. 11.The fine struc.
ture of Descement's membrane. J. Biophys. Biochem. Cytol. Suppl., 2: 243-252.
Kizior, J. E., J. W. Cuozzo and D. C. Bowman 1968 Functional and histologic assessment of the sensory innervation of the periodontal ligament of the cat. J. Dent. Res.,
47: 59-64.
Lewinsky, W.,and D. Stewart 1937 The innervation of the
periodontal membrane of the cat, with some observations
on the function of the end-organs found in that structure. J. Anat., 71:232-235.
Loewenstein, W.R. 1960 Biological transducers. Sci. Am.,
203: 98.108.
Munger, B.L. 1971 Patterns of organization of peripheral
sensory receptors. In: Handbook of Sensory Physiology.
Vol. I. W. R. Loewenstein, ed. Springer Verlag, Heidelberg-New York, pp. 523-556.
Ness, A. R. 1954 The mechanoreceptors of the rabbit mandibular incisor. J. Physiol. (London), 126: 475-493.
Patrizi, G.,and B. L. Munger 1965 The cytology of encap
sulated nerve endings in the rat penis. J. Ultrastruct.
Res., 13: 500-515.
Rapp, R., W. D. Kirstine and J. K. Avery 1957 A study of
neural endings in the human gingiva and periodontal
membrane. J. Can. Dent. Assoc., 23: 637-643.
Tandler, B., and C. L. Hoppel 1972 Mitochondria.
Academic Press, New York-London.
1 Sagittal section of t h e lingual part of the periodontal ligament. A, alveolar compartment; b, alveolar bone; d, dentin; T, tooth-related part. Hematoxylin and eosin. X 400.
2 Sagittal section of the alveolar compartment of t h e periodontal ligament. Nervous tis-
sue elements (ne) and associated K-cells (arrows) can be seen. Methylene blue.
3 Sagittal section of the alveolar compartment of the periodontal ligament. An
unmyelinated axon originates from a myelinated nerve fiber (arrowhead). Note the
kidney shape of the nucleus in the K-cells (arrows). Methylene blue. X 700.
V. Everts, W. Beertsen and A. van den Hooff
4 K-cell in close apposition to a nerve ending h e ) containing numerous mitochondria.
Where the cell extensions form a sheath around the nerve ending, layers of lamellar
material can be seen (arrowheads).bl, basal lamina; c, collagen. X 14,000.
V. Everts, W. Beertsen and A. van den Hooff
5 K-cell in close apposition to a nerve ending b e ) . bl, basal lamina; c, collagen.
x 20,000.
V. Everts, W. Beertsen and A. van den Hwff
6 Detail of the cell body of a K-cell. Note the presence of a Golgi apparatus (g).
7 Pair of centrioles with associated cilium extending from the cell body of a K-cell.
Note that many microtubules radiate from the centriolar region into the peripheral
cytoplasm (arrows). c, collagen; ne, nerve ending. X 15,000.
8 Bundle of microfilaments (arrowheads) in the peripheral cytoplasm of a K-cell. c,
collagen. X 37,000.
9 K-cell containing a leptomeric organelle (arrow) in the alveolar compartment of the
periodontal ligament. X 7,500.
10 Leptomeric organelle underneath the plasma membrane of a K-cell. Note the typical
cross-banding of the structure with a periodicity of about 160 nm. X 44,000.
V. Everts. W . Beertsen and A. van den Hooff
11 Axoplasmic extensions (ae) from the terminal region of a nerve fiber projecting
through small gaps of the cytoplasmic sheath. Note the presence of microfilaments
(mf) and vesicles. ce, cytoplasmic extension of a K-cell. X 35,000.
12 Junction between cytoplasmic extensions of K-cells surrounding a nerve ending h e ) .
X 46,000.
13 Giant mitochondrion in a nerve ending.
14 Giant mitochondrion in a nerve ending. Note the cross-sections of tubular infoldings
of the inner mitochondria1 membrane (arrows). ce, cytoplasmic extension of a K-cell
surrounding the nerve ending; lm, envelope of lamellar material consisting of
alternating layers of electron dense and electron transulcent material. X 19,000,
15 Crystal-like inclusions (arrows) in mitochondria of a nerve ending. ce, cytoplasmic
extension of a K-cell surrounding the nerve ending. X 31,000.
V. Everts, W. Beertsen and A. van den Hooff
16 Cytoplasmic extensions (ce) and a K-cell (kc) are surrounded by envelopes of
lamellar material (arrowheads). ne, nerve ending. X 6,000.
17 K-cell (kc) in close apposition to a nerve ending he). At the interface of basal lamina
and connective tissue cross-banded aggregates of filamentous material can be seen
(arrowheads). Note the presence of mitochondria (m) with elongated crystal-like
structures. c, collagen. X 12,000.
18 Bundle of filamentous material with a 100-150nm cross-banding. X 16,000.
V. Everts, W. Beertsen and A. van den Hooff
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structure, end, periodontal, mouse, ligament, incisors, organy, fine
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