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Microtubule and microfilament populations of cell processes in the dental pulp.

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THE ANATOMICAL RECORD 198:421-426 (1980)
Microtubule and Microfilament Populations of Cell
Processes in the Dental Pulp
(2. R. HOLLAND
Departments of Anatomy and Oral Biology, Colleges of Medicine and Dentistry,
llniuersity of Manitoba, Winnipeg, Manitoba R3E OW3
ABSTRACT
An attempt has been made to characterize the nature of the
unidentified cell processes participating in gap junctions in the odontoblast
layer. In peripheral and pulpal nerves, there is a strong relationship between
axon caliber and microfilament and microtubule populations. This characteristic,
together with the ratio of microtubules to microfilaments, has been measured
and compared for four types of cell processes found in the dental pulp, including
those participating in gap junctions. The processes taking part in the gap
junctions cannot be distinguished from pulpal axons on the basis of microtubuleto-microfilament ratio nor on the relationship between microtubule and microfilament population and process caliber. While these findings do not prove that
the "gap members" are nerve fibers, it does support the hypothesis that the
processes taking part in gap junctions in the peripheral dental pulp are nerve
fibers.
It has been suggested that some nerve fibers
in the dental pulp are electrically coupled
(Matthews and Holland, '75). Electrophysiological experiments strongly support this hypothesis (Matthews, '771, but, as yet, the anatomical basis of this coupling has not been
established. Gap junctions that occur predominantly in the odontoblast layer often involve
cell processes which may be axons (Holland,
'75, '76, '77). These gap junctions may couple
axons directly or through some intermediate
cell, possibly a n odontoblast. The principal
problem in testing this hypothesis has been
the difficulty in identifying, with certainty,
axons t h a t have lost their Schwann cell
sheath, a difficulty that also occurs in other
tissues (Whitear, '60; Munger, '66; Fillenz and
Woods, '70).
Friede and Samorjaski ('70) showed that in
the sciatic nerve of rats and mice, there was
a definite relationship between axon caliber
and neurofilament and microtubule populations. Bueltman et al., ('72) showed a similar
relationship for the pulpal axons in the marmoset. It may be that this relationship is
characteristic of axons. This paper reports an
examination of the possibility of using this
relationship to establish the axonal or other
nature of the cell processes taking part in gap
junctions in the peripheral pulp. To this end,
the microfilament and microtubule populations of three groups of cells, one definitely
0003-276)3180/1983-0421$01.400 1980 ALAN R. LISS, INC.
axonal and the others of connective tissue
cells, have been compared with the same feature of the unidentified cell process taking
part in the gap junctions.
MATERIALS AND METHODS
Three cats 6-8 months of age were anesthetized with sodium pentobarbitone and perfused via the left ventricle with a solution of
2.5% glutaraldehyde in 0.08 M phosphate
buffer for 1 hour. The canine teeth were removed and a disc 1 mm thick was cut from
the crown 4-5 mm from the tip of the tooth.
These discs were then washed in 0.08 M phosphate buffer and postfixed in 2% osmium tetroxide in distilled water for 2 hours. Following a wash in distilled water, the tissue blocks
were stained en bloc with 2% aqueous uranyl
acetate for 1hour. The blocks were dehydrated
in alcohols to which WOuranyl acetate was
added, except the absolute alcohol, and then
embedded in araldite resin. Ultrathin sections
of the odontoblast layer and central pulp were
cut and collected on formvar-supported slot
grids and stained with saturated aqueous urany1 acetate and lead citrate. The sections
were examined in the electron microscope at
~10,000.Micrographs were printed at ~30,000
of four types of cell processes:
1) Processes in the odontoblast layer which
Received September 11, 1979 accepted February 29, 1980.
422
G. R. HOLLAND
participate in gap junctions (Fig. 1).
2) Odontoblastic processes in the predentin
(Fig. 2).
3 ) Non-myelinated axons in the sub-odontoblastic region (Fig. 3 ) .
4) Processes in the central pulp, presumably
fibroblastic (Fig. 4).
Many processes from each group were photographed from each specimen, with no attempt a t selection. After printing, those processes which were outside the caliber range of
non-myelinated axons in the pulp established
by Beasley and Holland ('78) were eliminated,
as were those processes in which the microtubule and microfilaments could not be counted due to obliquity.
The profiles of the axons and cell processes
were traced on acetate sheets and their area
measured on a Quantimet Image Analysis
System. The number of microtubules and microfilaments within each axon and process was
counted.
The two features that showed the strongest
correlation in Friede and Samorjaski's ('70)
study were the microfilament-to-microtubule
ratio and the relationship between process
caliber and total population of microfilaments
and microtubules. These features were compared for all four groups of processes.
STATISTICAL TREATMENT OF DATA
The ratios of microtubules to microfilaments
for each group were compared with the others.
The homogeneity of the variances was first
tested for each pair of samples using Bartlett's
test (Brownlee, '65). If the variances were
homogeneous, the comparison was made using
the student t-test. If the variances were not
homogeneous, Welch's approximation was applied and a modified student t-test used
(Brownlee, '65).
The relationships of microfilaments and microtubule population to process area for each
group were also compared with each of the
others. These relationships were estimated by
regression lines. If the variances proved homogeneous by Bartlett's test, a n analysis of
covariance (Dunn and Clark, '74) was performed and the slopes of the regression lines
compared. If the variances were not homogeneous, Welch's approximation was employed,
combining the variances, and the modified ttest used to compare the slopes. If slopes were
not significantly different, intercepts were also
tested for differences.
RESULTS
1) Microtubule-to-microfilament ratios
These are shown in Table 1. Table 2 shows
the result of the comparison of the different
groups of processes.
2 ) Microtubule and microfilament population
related to process caliber
Slopes, intercepts, and correlation co-efficients of this relationship are shown in Table
3 . The regression lines themselves are shown
in Fig. 5. Tables 4 and 5 show the comparison
of these regression lines.
DISCUSSION
The relationship between axon caliber and
tubule and filament numbers is as strong for
pulpal axons (correlation coefficient 0.70) as
Friede and Samorjaski ('70) (c.c. 0.78) demonstrated for axons in the sciatic nerve. The
slope on the regression lines is, however, different from that for non-myelinated axons in
the sciatic nerve (Friede and Samorjaski, '70)
but is surprisingly similar to that for myelinated nerves. Similarly, the microtubule:
microfilament ratio for unmyelinated axons in
the pulp is almost identical to that for myelinated axons in the sciatic nerve. The reason
for this similarity is that the pulpal axons are
probably the preterminal branches of myelinated axons and have retained their characteristics. Few non-myelinated axons are found
near the apex of the tooth, often fewer than
400 (unpublished observation),yet at the midcrown level, several thousand are found (Beasley and Holland, '78). Other workers have also
reported the preponderance of microtubules
over microfilaments in non-myelinated pulpal
axons (Stockinger and Pritz, '71; Bueltman et
al., '72).
There is also a good correlation between
process caliber and microtubule and microfilament population for the processes taking
part in the gap junctions (correlation coefficient 0.71) and for the fibroblast process of the
central pulp (correlation coefficient 0.84). The
correlation is low (correlation coefficient 0.28)
for odontoblastic processes in the predentin.
When the four types of processes are compared, it is not possible to distinguish between
pulpal axons and the processes taking part in
gap junctions on the basis of their microtubule
to microfilament ratios. It is, however, possible
to distinguish between the "gap members"
CELLPROCESSESOFTHEDENTALPULP
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
A typical gap junction from the odontoblast layer. x 45,000.
An odontoblast process from the predentine. x 45,000.
A pulpal axon ensheathed in a Schwann cell. X 45,000.
A fibroblast process from the central pulp. x 45,000.
423
G.R. HOLLAND
424
TABLE 1 . Microtubule-bmicrofilament ratio for cell processes in the
dental pulp and predentin.
No. of
orocesses
Mean
microtubule-tomicrofilament ratio
S.D.
22
0.22
0.28
14
21
9
0.24
0.10
1.70
0.18
0.13
0.91
Processes participating
i n gap junctions
Pulpal axons
Fibroblast processes
Odontoblast processes
TABLE 2. Comparison of microtubule and micmfilament ratios for cell
processes in the dental pulp and predentin.
Gap members v. axons
Odontoblast pr. v. axons
Fibroblast pr. v. axons
Odontoblast pr. v. gap
members
Fibroblast pr. v. gap
members
Fibroblast pr. v. odontoblast
pr .
D.F.
t
Significance level
35
8.404
33
8.637
-0.74
-4.689
3.10
-4.787
N.S.
< 0.01
< 0.01
< 0.01
1.75
< 0.05
-5.239
< 0.01
41
8.14
TABLE 3. Regression analysis of microtubule and microfilament counts with pmcess
caliber.
Slope of regression
Process participating
in gap junctions
Pulpal axons
Fibroblast processes
Odontoblast processes
Intercept
Correlation coefficient
221.436
86.8638
0.71
352.895
215.86
49.24
-0.0754
29.4846
119.029
0.70
0.84
0.28
TABLE 4. Comparison of regression lines.
Slope
F.
-.
Gap members
vs. pulpal
axons
Gap members
vs. fibroblast
pr .
Pulpal axons vs.
fibroblast ur.
-
(D.F.)
Intercept
~~
Signif.
F.
. . ~ _ _ _ _ _ . _ - -
(D.F.)
Signif.
~~
1.265
(1,321
NS
2.503
(1,33)
NS
0.009
(1,39)
NS
9.935
(1,40)
0.01
1.94
(1,31)
NS
1.214
(1,321
NS
TABLE 5. Comparison of regression lines by t-test after Welch's approximation.
_
~-
__
Gap members vs.
odontoblast
processes
Pulpal axons vs.
odontoblast
processes
Fibroblast pr. vs.
cdontoblast
Drocesses
_
T
_____
Slopes
_
_
_
~
~
(D.F.)
Significance
-7.3
(12.83)
< 0.01
-8.69
(20.977)
< 0.01
(9.814)
< 0.01
-7.491
Intercepts
_ ~ _ . . ~ _ _ . _ _ _ _ _
Comparison
not valid, as
slopes differ.
425
CELL PROCESSES OF THE DENTAL PULP
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426
G. R. HOLLAND
and odontoblast and fibroblastic processes.
When the same comparisons are made using
the caliber-to-population relationships, both
the slopes and intercepts of the gap members
and pulpal axons are not significantly different. The gap members do differ significantly
from both the fibroblast and odontoblastic
processes on this basis, although using this
feature, it is not possible to recognize pulpal
axons and fibroblasts as different populations.
The lack of difference between pulpal axons
and fibroblastic processes may be important.
It may, however, be due to the “contamination” of the fibroblastic population with unsheathed axons. The fact that regression lines,
except in the case of the pulpal axons, do not
pass through zero is interesting. In the case of
the gap members, the odontoblast processes,
and the fibroblast processes, it presumably
indicates that there is a minimal number of
these structures always present, even in the
narrowest of processes. In the case of the
pulpal axons, the intercept is close to zero, but
statistically, this was not significantly different from the intercept for the gap members.
The principal conclusion from the findings
must be that the processes taking part in the
gap junctions in the peripheral dental pulp
cannot be distinguished from pulpal axons on
the basis of their microtubule and microfilament populations. Several earlier workers
have attempted to identify structures involved
in specialized junctions in the pulp and dentin
as nerves (Stockinger and Pritz, ’70; Vacek
and Palckova, ’59; Arwill, ’68; Frank, ’66a, b,
’68a, b). They used only qualitative morphological criteria, and their conclusions have not
achieved widespread acceptance. While the
current findings do not prove that the “gap
members” are nerve fibers, it does support the
hypothesis quantitatively and suggests that
other techniques be pursued to test the hypothesis further.
ACKNOWLEDGMENTS
The data reported in this paper was collected while the author was a member of the
Department of Endodontics and Dows Institute, College of Dentistry, University of Iowa.
I would like to thank Doug Holmes, who
helped with microscopy, and Barry Rittman,
who carried out the measurements on the
Quantimet System.
The statistical analysis was conducted by
Mary Cheang of the Computer Department
for Health Sciences, University of Manitoba.
This work was supported by USPHS grant
5-501-5313 and grant Number 321-3113-04
from the Medical Research Council of Canada.
LITERATURE CITED
Arwill, T. (1968) The ultrastructure of the pulpo-dentina1 border zone. In: Dentine and Pulp: Their Structure and
Reactions. N.B.B. Symons, ed. Livingston, London pp.
147-167.
Beasley, W.A., and G.R. Holland (1978) A quantitative
analysis of the innervation of the pulp of the cat’s canine
tooth. J. Comp. Neurol., 178.487-494.
Brownlee, K.A. (1965) Statistical theory and methodology in science and engineering, 2nd Ed. Wiley, N.Y.
Bueltmann, K., U.L. Karlsson,and J.Edie (1972) Quantitative
ultrastructure of intradental nerve fibers in marmosets.
Archs. oral Biol., I7:645-660.
Dunn, O.J., and V.A. Clark (1974) Applied Statistics:
Analysis of Variance and Regression, Wiley N.Y.
Fillenz, M., and R.J. Woods (1970) Sensory innervation
of the airways. In: Ciba symposium: Breathing, HeringBrauer Centenary Symposium. R. Porter, ed. Churchill,
London, 101-107.
Frank, R.M. (1966a) Etude a u microscope electronique
de I’odontoblast e r du canlicule dentinaire humain.
Archs. oral Biol., 13:833-834.
Frank, R.M. (1966b) Ultrastructure of human dentine.
In: Calcified Tissues 1965. H. Fleisher, H.J. Blackwood,
and M. Owen, eds. Springer-Verlag, Berlin. pp. 259-272.
Frank, R.M. (1968a) Ultrastructural relationship between the odontoblast, its process and the nerve fibre. In:
Dentine and Pulp: Their Structure and Reactions. N.B.B.
Symons, ed. Livingston, London. pp. 115-145.
Frank, R.M. (1968b) Attachment sites between the odontoblast process and the intradental nerve fibre. Archs
oral Biol., 13833-834.
Friede, R.L., and T. Samorjaski (1970) Axon caliber related to neurofilaments and microtubules in sciatic nerve
fibers of rats and mice. Anat. Rec., 167:379-388.
Holland, G.R. (1975) Membrane junctions on cat odontoblasts. Archs. oral Biol., 20:551-552.
Holland, G.R. (1976) Lanthanum hydroxide labelling of
gap junctions in the odontoblast layer. Anat. Rec.,
186: 121- 126.
Holland, G.R. (1977) Structural relationships in the
odontoblast layer. IN: Pain in the trigeminal region. D.J.
Anderson and B. Matthews, eds. ElsevieriNorth Holland,
Amsterdam. pp. 25-35.
Matthews, B. (1977) In: Pain in the Trigeminal Region.
D.J. Anderson and B. Matthews, eds. ElsevieriNorth
Holland, Amsterdam.
Matthews, B., and G.R. Holland (1975) Coupling between nerves in teeth. Brain Res., 98: 354-358.
Munger, B.L. (1966) Discussion in: Touch, Heat and
Pain. V.S. de Reuck and J. Knight, eds. Churchill, London. p. 129.
Stockinger, L., and Pritz, W. (1970) Morphologische Aspekte der Schmerzempfinding im Zahn. Dt. Zahn.,
2(25):557-565.
Vacek, Z., and H. Palckova (1959) Die innervation der
zahne. Acta. Anat., 36:59-77.
Whitear, M. (1960) An electron microscope study of the
cornea in mice, with special reference to the innervation.
J. Anat., 94:387-409.
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