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Dentin apposition rates as markers of primate growth.

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AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 55:443 -453 (1981)
Dent n Apposition Rates as Markers of Primate Growth
S. MOLNAR. T.R. PRZYBECK.
i ~WILKERSON
.
D.G. GANTT. R.S. ELIZONDO. AND
Washington University, St. Louis, Missouri 63130 (S.M., T.R.P.);
Florida State
University, Tallahassee, Florida (0.
G.G.); and Indiana University,
Bloornington, Indiana (R.S.E.,J.E.W.)
KEY WORDS
lines
Primate growth, Primate teeth, Dentin, Incremental
ABSTRACT
The incremental lines of von Ebner frequently have been described as ultradian markers of dentin calcification. To determine the relationship
between these lines and the quantity of dentin formed, reference was made to an in
vivo marker of calcification, tetracycline. These markers were produced by injecting four juvenile rhesus monkeys periodically over a period of 175 days. These
animals had been subjects in a heat stress study and were exposed to a series of
heat stresses and cortisone injections. A t the end of the study, undecalcified thin
sections of premolars and second molars were prepared by standard histological
techniques.
We found that linear apposition rates increased in a gradient toward the pulp
chamber. These rates varied within each tooth but not in corresponding parts of
different teeth. Also, we determined that dentin apposition can be temporarily
depressed by certain metabolic stresses. Heat load and cortisone significantly
slowed dentin formation. Each depression, however, was followed by a "catch-up''
period. Incremental line distances also increased as a function of the distance from
the dentoenamel junction. There was, however, no systematic relationship between apposition rates and incremental line distance; these distances did not
deviate from the observed trends during periods of slowed apposition. Incremental
lines may be interpreted, not as natural growth markers, but as structural
phenomena which are a function of dentin geometry and tubule bending.
The incremental nature of tooth development has attracted the attention of researchers
for many years. I t has been assumed that the
apposition of the organic matrix and its mineralization proceed at a rate reflective of the
biological rhythm of the organism. It is
thought that disruptions of this rhythm,
through metabolic depressions that affect
mineralized tissues, should be indicated by
structural changes that are visible through
light microscopic examination of undecalcified
thin sections. Dentin, in particular, has been
described as especially prone to faulty calcification and is considered a more sensitive indicator of mineral metabolic fluctuation than
enamel (Sognnaes, '61:727; Schour, '38:53).
Faulty calcification may appear microscopically as interglobular dentin or as broad
hypomineralized bands, which are referred to
as Contour Lines of Owen (see Sicher and
Bhaskar, '72109).
0002-948318115504-0443$03.50
0 1981 ALAN R. LISS, INC.
Another feature of dentin which has received
a great deal of attention is the alternating pattern of light and dark lines, the incremental
lines of von Ebner (Fig. 1).These incremental
lines, which represent alteration in the organization of the matrix, are presumed to be a
record of dentinal apposition rates and constitute markers of circadian or ultradian rhythm
(Schour and Massler, '40). The space between
these lines has been measured in several mammalian species and the distances were reported
to be 16 microns (pm)(seeSchour and Hoffman,
'39a, b). Though this distance appears to be
relatively constant between species, the time
interval between lines varies considerably.
Yilmaz et al. ('77) used lead acetate as an in
vitro marker in the teeth of domestic pigs and
Received September 10,1979: accepted February 13, 1981
444
S. MOLNAR ET AL.
Fig. 1. Longitudinal undecalcified section of a lower second premolar of a rhesus monkey viewed under bright light
microscopy to reveal incremental lines (original magnification 100 X).
reported that there were 13 sets of alternating
light and dark lines between markers injected
at 2-week intervals. This indicated that each
set of incremental lines represented a circadian
unit of apposition. Calculations of dentinal apposition rates by reference of incremental lines
to in vivo markers such as a alizarin and
sodium fluoride were made by Schour and his
co-workers, who reported that dentin is
deposited at the rate of 4 pmlday in man and
rhesus and 16 pmlday in rodent, cat, and dog.
The interval between lines, 16 pm, was distinguished as a 4-day episode in man and rhesus
but only a single day in the other species
(Schour and Hoffman, '39b).
In an earlier study, Schour and Poncher ('37)
reported an apposition rate of 3.77 pmlday over
a 115-day period in the central incisor of a
human infant with inoperable hydrocephalus
and meningocele. They used sodium fluoride
to delineate 20 growth intervals, and for these
intervals apposition rates varied from 3.16 pm
to 4.42 pmlday. They did not, however, note
any systematic trends in the variation of these
rates. Schour and Hoffman ('39b)found dentin
apposition rates of between 3.68 pm and 4.40
pmlday in the gingival third and roots of
premolars and molars in a series of 17 rhesus
macaques. These animals ranged in age from
1.5 to 7.0 years. Though the authors gave no indication that the variation in rates was related
to the ages of the animals, they noted gradient
effects at different levels in a tooth. A rate of
12.2 pmlday was found in the cuspal portion of
the third molars, and a rate of 2.4 was found at
the apical level of the canines and premolars.
Thus it is clear that this early work, while indicating an average rate of dentin apposition of
4 pmlday, does not demonstrate that apposition proceeds at a uniform rate as has been commonly assumed and stated. Nonetheless, the
conclusions from these studies have become incorporated in many standard oral histology
texts and the 4- and 16-pmintervals are usually
accepted as indicative of biorhythm, and the incremental lines are frequently described as
useful markers of time intervals (Sicher and
Bhaskar '72:108; Symons, '67:311).
However, statements about constant growth
rate have not gone unchallenged. Kraus ('59)
DENTIN APPOSITION RATES AS MARKERS OF PRIMATE GROWTH
noted the discrepancy between tooth apposition rate and the calculated growth rates of
other body tissues if the 4- and 16-pmintervals
were accepted as a standard for tooth formation. He showed, through measurements of
enamel of deciduous teeth from 76 human
fetuses, that all areas of a tooth do not calcify a t
a uniform rate and that growth proceeds more
rapidly in some dimensions than in others; for
example, mesiodistal diameter increases faster
than cusp height. Further, when any one tooth
dimension is plotted against age (in weeks),the
regression is curvilinear indicating changing
velocity in the calcification process.
More recently, Kawasaki et al. ('77)reported
that the rate of dentin formation changes continually. Their study of a series of human
premolars and molars taken from individuals
receiving therapeutic doses of tetracycline
demonstrated that the rate of apposition slowly increases during the first third of dentinogenesis. During the second third the rate is
relatively higher, and during the final third the
rate declines steadily. Also, Melsen et al. ('77)
reported that before eruption an apposition
rate 3 1 pm per day is found in the teeth of
both humans and Macaca mulatta. This rate
decreases at eruption in the region over the
pulp chamber but not in the roots. Finally,
Rosenberg and Simmons ('80) examined incremental lines in the continually growing incisors of the rabbit and found that in decalcified sections of different thickness incremental
lines of different widths were observable in-the
dentin. Ebctron microprobe scans of calcium
and sulfur distributions, indicative of the density of the mineral and organic phases, respectively, failed to reveal any systematic relationship between the pattern of the incremental lines and variation in calcium or sulfur
distribution. The authors concluded that incremental lines are not a natural marker of
growth but rather a structural phenomenon.
These demonstrations, of course, raise
questions about the nature of the rate of apposition and about the use of incremental lines
as growth markers. In our study we have attempted to clarify the relationship between incremental lines and dentin apposition. Do the
incremental lines demark episodes of growth?
Is their spacing regular throughout the dentin?
Also, can these naturally occurring lines be
used to establish agrowth rhythm which would
mark a normal pattern or identify depressed
rates?
*
MATERIALS AND METHODS
In order to examine the relationship between
dentin apposition rates and the spacing of in-
445
cremental lines we studied teeth from two
groups of rhesus monkeys (Macaca mulatta).
One group was labeled with an in vivo marker,
which permitted the measurement of apposition rates and incremental line spacing (Figs. 1
and 2). The second group was not labeled.
Measurements of incremental lines in this
group were used to assess possible effects of
labeling on the incremental lines in the other
group.
Labeled group
This group of four juvenile male rhesus, approximately 1.5 years old, were injected at intervals with tetracycline (6 mglkg, intravenously) over a 175-dayperiod in order to provide in vivo markers of dentin development.
Tetracycline has proved effective as a marker
of calcifying tissue and, at normal dosages,
does not affect dentinogenesis according to recent reports by Yen and Shaw ('74) and
Kawasaki ('75).Binding with the dentin, tetracycline indicates the incremental pattern of
growth of this tissue and can be seen under
ultraviolet microscopy. Figure 2 shows the
typical pattern of fluorescent banding provided by tetracycline labeling.
The animals were part of a thermoregulation
study. Each was subjected to three short
periods (60-90 minutes) when it was exposed to
ambient temperatures of 100-110°F at 20%
humidity in a climatic chamber while held in a
restraining chair. During these periods rectal
temperatures increased from 3" to 5" above
normal. On the 88th and 155th day of the study
there were two longer heat stress periods. All
four rhesus were kept in the climatic chamber
for 48 hours at 100°F beginning on the 88th
day and for 31 hours at 110°Fbeginning on the
155th day.
In addition to the heat stress periods, two
series of cortisone injections were given to determine its effect on dentin apposition. I t has
been previously reported that cortisone inhibits bone growth and may also influence dentin apposition (Follis, '51; Moss, '55; Frost, '73).
An injection of 4 mg of dexamethasone was
given on each of 3 days beginning on the 125th
day. Tetracycline markers were injected before
and after this period. The dexamethasone injections were repeated for 5 days beginning on
the 164th day.
These physiological stresses were given with
the intent of inducing disruptions in the normal
pattern of dentinogenesis. Our initial expectations were that anomalies of development such
as interglobular dentin (areas of hypocalcifica-
446
S. MOLNAR ET AL
Fig. 2. Same field as shown in Figure 1. viewed under ultraviolet light demonstrating tetracycline labels.
tion) might be associated with these disruptions and that the tetracycline markers would
serve to identify the stress responsible for the
observed anomaly. The stresses produced several problems with respect to the interpretation of apposition rates; this notwithstanding,
a clear pattern emerged, indicating a resilience
in the forming dental tissue.
After 175 days the animals were terminated.
The teeth were removed and prepared for histological analysis by standard methods. The
premolars and second molars were extracted
(12 teeth from each animal) and, after cleaning
and drying in alcohol and acetone, were embedded in an epoxy resin. Undecalcified thin
sections of 80-100 pm were made with a Gillings thin section diamond saw. The teeth were
cut longitudinally in the buccolingual plane
through the mesial cusps.
After mounting on a glass slide under a
coverslip, each section was examined under
light microscopy for evidence of incremental
lines and Contour Lines of Owen. When groups
of incremental lines were located photographs were made at 1OOx magnification
with Ektachrome film (ASA 125). The microscopic field was photographed again under ultraviolet light at the same magnification to
reveal the fluorescent bands of tetracycline.
This procedure was repeated for each area for
each section where incremental lines were
distinct. A photograph of a stage micrometer
(2-mmscale) was made at the same magnification for calibration. The microphotographic
slides (35-mmEktachrome)were projected by a
Kodak carousel projector (50-mmlens) from a
measured distance onto a specially prepared
translucent screen. The 18" X 27" image permitted the easy measurement of the incremental line spacing from the reverse side of the
screen with high precision (less than 3% difference in repeated measurements). The measurements were converted to microns by comparison to the stage micrometer image (a conversion of 1mm = 1.4 pm).This method provided more precise results than had been achieved
by direct measurement of the thin section with
a filar micrometer, which produced errors of
10-12%. The distance between incremental
lines was measured according to the method of
DENTIN APPOSITION RATES A S MARKERS OF PRIMATE GROWTH
Schour and Hoffman ('39a) - that is, from the
beginning of one dark band to the beginning of
the next along the path of the dentinal tubules.
The measurements of the tetracycline bands
made by this method compared well with those
previously obtained from enlargements of
black and white photographs. The tetracycline
bands were more regular and uniform in certain
regions of each tooth section. In the 48 sections
(12 from each animal) on the buccal and lingual
sides, approximately half way between the dentinal horn and the cementoenamel junction
(CEJ)was clearest. We refer to these areas as
regions A2 and B2, respectively (see Fig. 3).
These data comprise a large number of measurements which demonstrate clear patterns.
For comparative purposes a similar, but much
smaller, data set was generated for the region
between the dentinal horns, the AB region,
where the banding was less clear. While this
smaller set of data does not, by itself, permit
conclusions to be drawn, it does support the
other data and suggests that the same trends
exist in all parts of the tooth.
The spacing of the tetracycline band groups
over the 175-day study bracketed 13 intervals,
and the distance between each marker was
measured. The rate of dentin apposition during
each interval was determined separately for
each tooth and area in each animal.
We determined the rates of dentin apposition
in different parts of the teeth and measured the
influences of the applied stresses on apposition
rates. The distances between incremental lines
were established and the two sets of data were
compared to clarify the meaning and nature of
incremental lines as natural markers of
dentinogenesis.
Unlabeled group
This group consisted of four adult animals
that had not been injected with tetracycline.
Eight teeth from these animals were sectioned
and photographed as described above. Measurements were made of the incremental lines
in the A2 and AB regions. These measurements were compared to those of the labeled
group to insure that incremental line distances were not influenced by the tetracycline
injections.
447
Fig. 3. Schematic of tooth section. Buccal side of the
tooth is to the right. The areas labeled A2. B2, and AB are
those from which all measurements were taken,
analysis of variance ( F = 2.98, P > 0.05) indicated that it would be appropriate to pool
the measurements from the A2 and B2 areas
in the four animals. This produced a data set
consisting of up to 96 measurements per band
interval, consisting of two measurements
from each tooth, one buccal and one lingual, in
a total of 1 2 teeth from each animal. Because
some sections did not have all tetracycline
bands clearly defined, the actual number of
measurements for each interval ranged from
41 to 82. The results of these measurements
are summarized in Table 1. The apposition
rates in the A2 and B2 regions show a distinct
temporal trend. During the first interval,
which begins 300 pm from the dentoenamel
junction (DEJ),the apposition rate is 2.91 pml
day. Subsequent intervals show a steady inRESULTS
crease until in the final interval a rate of 3.91
pmlper day is reached (Fig. 4). The rate of apApposition rates
position averaged over the 175-day period is
Comparisons of all teeth by class and by 3.27 pm per day, which is similar t o the
area of measurement showed a greater than averages reported by several previous in0.9 c o r r e l a t i o n coefficient b e t w e e n vestigators.
The small set of measurements from the AB
tetracycline band intervals in isomeres and antimeres for each area. Q-mode correlation and region (the intercuspal area) indicate that ap-
448
S. MOLNAR ET AL.
TABLE 1. Mean daily rates of apposition (labeled animals)
Tetracycline
interval
1 2
2
33
4
5
6
7
83
9
10
113
123
134
Length of interval
(days)
N'
A2 and B2 area
17
22
16
18
22
22
12
4
10
7
3
15
7
67
78
80
82
82
81
78
75
74
66
62
60
41
AB area
-
XIpm)
S.D.
N
X(&m)
S.D.
2.91
2.71
3.10
3.27
3.26
3.50
3.52
3.22
3.92
3.57
3.34
3.75
3.91
0.575
0.459
0.572
0.542
0.529
0.547
0.567
0.600
0.687
0.597
0.770
0.604
0.512
2
4
6
6
7
5
2
1
1
1
2.87
2.90
3.86
4.52
4.54
4.91
4.58
5.37
4.27
4.66
0.665
0.273
0.900
1.05
0.983
1.03
0.375
-
-
'Based on measurements from buccal and lingual areas in 12 teeth from each of four animals IN,,,
2300pm from DEJ.
31ntervalsduring which metabolic stress was induced. See text for discussion.
'875 um from DEJ.
-
-
-
= 961.
Y-2.75 + .006X
/
r = ,934 (P < ,011
3.5
I
A
Heat 1
Cortisone 1
A
0 No stress
25
0
Heat 2
Cortisone2
,
I
1
1
I
I
30
60
90
120
150
180
TIM€ (DAYS)
Fig. 4. Plot of average apposition rates from A2 and B2 areas combined. Episodes of applied stress are indicated. hut were
not used to calculate regression. Each point is at the midpoint of a tetracycline interval. Values are presented in Table I . See
text for discussion.
the animals were maintained for 31 hours at
110°F at the beginning of a 3-day interval
(heat 11). The first heat episode (heat I) was
longer (48 hours), b u t t h e ambient
temperature was 10°F less. Possibly, the
rhesus were better able to thermoregulate at
100" than at higher temperature. At least the
metabolic processes suffered less disruption
Response to stress
as evidenced by the dentin rate during inHeat stress, applied over several hours, terval three (heat I). This distinction between
caused a reduction in dentin apposition. This the two heat episodes may prove to be of
was recorded during the 11th interval when significance and suggests a threshold in the
position rates are, on the average about 1 pmlday greater than in the A2 or B2 regions.
Finally, the teeth we examined were in a stage
of development which corresponds to the second third of development as described by
Kawasaki et al. ('77), and conform to their description of relative rates.
449
DENTIN APPOSITION RATES AS MARKERS OF PRIMATE GROWTH
sion of apposition rate would be averaged out
by the “rebound effect,” which again indicates that without strategically placed in vivo
markers it may be extremely difficult to discern depressions or disturbances of dentin
growth since the tissue apparently has the
ability to compensate for these depressions by
later accelerated apposition.
None of the stress periods induced the gross
imperfections in dentin known as interglobular
dentin (IGD).This lack of IGD in the rhesus
teeth is consistent with previous reports on
primate dental histology and our own observations of samples from other macaques (Molnar
and Ward, ’75). But the failure to induce this
imperfection in the dentin matrix by heat
stress or cortisone treatment does not rule out
the possibility that a pyrogenic agent might
cause this defect.
sensitivity of the dentin matrix to metabolic
disturbances. The shorter heat stress periods
during intervals 5 and 6 did not affect dentin
development noticeably. The dentin apposition rate varied in the same way for all
animals, and a pattern of response is clear;
small increase in body temperature may cause
slight depressions in dentin apposition rate,
but these changes can only be detected if the
period is closely bracketed by in vivo markers
to exclude the “catch-up”period following this
interval. The lack of response seen in interval
3 (heat I) may be related to this, since the heat
strss fell at the beginning of the 22-day interVal.
The first point of significant depression is
seen during the second interval. No stresses
were applied during this interval, which
makes it difficult to explain the reduction in
apposition rate. A suggestion is offered that
the animals were still undergoing adjustment
to their new quarters. Frost (’69:216) cautioned that animals must be preconditioned in
the experimental environment for a time equal
to “one sigma.” How this period, the time it
takes for a basic bone unit to remodel, compares to dentin apposition rates we do not
know.
The first series of cortisone injections,
though in normal therapeutic concentrations,
caused detectable depressions in dentin apposition rates. Following the cortisone series a
period of accelerated growth is seen as a “rebound effect” similar to that found in humans
following treatment with corticosteroids (S.
Teitelbaum, ’77, personal communication).
The second period of cortisone (5 days a t
24-hour intervals) showed no response comparable to the first series. This may be due to the
long period between tetracycline injections at
this point in the study (15 days). Any depres-
Incremental lines
Interline measurements were made in three
regions of the teeth as was done with the tetracycline measurements. Analysis of variance
showed that there was no difference between
teeth (PM3, PM4, M2),so all teeth were pooled
as was done with the tetracycline band
measurements. The mean incremental line distances are listed in Table 2 for each of the tooth
regions, in correspondence to the tetracycline
band intervals, a position relative to the dentoenamel junction. The first tetracycline
marker is 300 p from the dentoenamel junction.
The distance between lines in area A2 are not
significantly different from comparable lines in
the B2 area, but the incremental lines in the AB
area are, on the average, farther apart. A total
of 516 incremental line measurements were
made on sections from the labeled animals. The
mean distance between incremental lines was
16.87 pm. The average is approximately 1 pm
TABLE 2. Incremental line distances for each area and group (labeled animals)
Area
~-
~~
Band intervals
1
2
3
4
5
6
7
8
9
10. 11,123
13
Total
4‘(2)’
10 (31
16 (6)
26 19)
49 (13)
47 (12)
31 113)
9 (6)
22 (11)
40 (91
10 (71
264 (14)
~~~~
82
A2
Mean
S.D.
13 76
15 04
15 70
14.99
15.21
15.33
16 72
17 34
17 29
17.98
17.74
16.15
1.OO
2.72
3.04
2.20
2.03
2.39
2.78
1.85
3.55
2.93
3.18
2.82
N
5 (1)
7 (2)
12(4)
13(4)
16 (6)
14(5)
8 (4)
4(4)
lO(5)
1814)
7 (3)
114(7)
Mean
SD
1596
1656
1561
1506
1498
1599
1727
1547
1763
1848
1744
1633
096
160
288
283
309
313
235
259
2 18
200
292
279
AB
Mean
~
SD
13.44
14.12
17.28
21.32
20.87
21.49
21.00
22.5
22.75
24.31
0.10
0.80
19.01
4.55
-
1.80
1.64
3.98
4.44
4.32
3.38
1.75
-
-
‘Number of incremental lines.
2Numher of teeth.
>Becauseintervals 10. 11, 12 were too short to include incremental lines in each. measurements were taken from the beginning of 10 to the end of
12.
450
S. MOLNAR ET AL.
greater than the mean of 15.85 pm reported by
Schour and Massler (’40) for the rhesus
monkey. A series of 144 incremental line
distances on the tooth sections of the unlabeled
animals is consistent with the data from the
labeled group, giving a mean of 16.81 pm.
The differences between our data and Schour
and Hoffman’s (’39a)are not readily explicable.
The most probable cause is that our measurements were taken from all regions of the teeth.
If we separate the measurements according to
region (aswith the tetracycline measurements)
(see Tables 2 and 3), it can be seen that the
means for the A2 and B2 regions in both the
labeled and unlabeled groups are very close to
Schour and Hoffman’s findings. The labeled
group has means of 16.15 and 16.37 pm for the
A2 and B2 regions, respectively; the unlabeled
group has a mean of 16.3 pm for the A2 region
(no B2 measurements were taken). In both
groups, however, the mean in the AB region is
larger, 19.01 in the labeled group and 17.36 in
the unlabeled group. Further, since Schour and
Hoffman used transverse sections for their
study, the level of sectioning may have limited
the variation of their measurements. This is
probable in light of the fact that we see a difference in the average interline distance in different regions of the same tooth. Although
there is no indication of the level at which they
made their sections, it is most likely that they
more closely approximate the level corresponding to our A2 and B2 regions in the cervical
rather than intercuspal region, since sections
here would not expose the pulp chamber if
taken transversely. As a result of these considerations, there appears to be no great disparity
in the results of the Schour and Hoffman study
and our results. The validity of an approximately 16-pm average distance between incremental lines in the cervical portion of the tooth
is confirmed.
While our average distance is in agreement
with previous work, we noticed that the variation around that mean is not random; in fact,
the lines are spaced farther apart near the pulp
chamber, and there is a high correlation between distance from the DEJ and distance between incremental lines. This relationship is
illustrated in Figures 5 and 6. Figure 5 is a plot
of incremental line distance in the A2 region
from the labeled group. Each point is the mean
incremental line distance for each of the tetracycline band intervals. This plot represents the
best fit of the four we calculated (A2 and AB
regions from both groups of animals). Figure 6
is a plot of the mean incremental line distances
A 2 A R E A (915)
19.0
~1
W
r z . 9 2 3 . P<.OI
W
a
I
13.01
Q
I
c
u)
6 1201
200
400
600
DISTANCE FROM 1st TC MARKER(y1
Fig. 5. Plot of average incremental line spacing in A2
area in labeled animals. The initial tetracycline label was 300
pm from the cementaenamel junction in this area of the
tooth. Values are presented in Table 2. See text for discussion.
TABLE 3. Incremental line distance for each area (unlabeled animals)
Area
Distance from
DEJ (pm)
0-100
100-200
200-300
300-400
400-500
500-600
600-700
700-800
800-900
Total
‘Number of incremental lines
lNumber of teeth.
A2
N
AB
Mean
S.D.
N
Mean
S.D.
-
-
14.78
14.57
14.54
15.23
17.20
19.10
23.81
24.71
16.37
1.35
2.80
1.84
1.59
3.36
2.46
1.67
1.41
3.63
9 (2)
12 12)
6 (21
7 (2)
9 (2)
8 (2)
13.55
15.39
18.32
18.77
16.05
19.01
18.19
22.19
22.88
17.36
2.09
3.38
6.37
4.27
2.54
3.48
2.97
0.60
1.62
4.14
6 12)
5 (1)
2 11)
64 (4)
451
DENTIN APPOSITION RATES A S MARKERS O F PRIMATE GROWTH
TABLE 4. Comparison of regression analysis in
labeled and unlabeled animals for relationship
between incremental line distance (Y) and
distance from DEJ (X)
A2
Labeled
Unlabeled
r = 0.916 ( P < 0.01)
.
100
'
400
+
+
--
Area
___
Band intervals
1
'
AB
Y = 13.93 0.06 X
r = 0.912 ( P < 0.01)
Y = 13.93 0.01 X
r = 0.872 (P < 0.01)
TABLE 5. Periodicity of incremental lines'
/
I3L
+ 0.01 X
r = 0.923 ( P < 0.01)
Y = 10.17 + 0.02 X
Y = 13.95
800
600
DISTANCE FROM DEJ
(PI
Fig. 6. Plot of average incremental line spacing in AB
area in unlabeled animals. Interline distances are grouped in
increments of 100 pm starting from the cementoenameljunction. Values are given in Table 3. See text for discussion.
in each 100-micron increment traversed from
the DEJ to the pulp chamber. This plot represents our poorest fit of the four, but the correlation is still high (r = 0.872). The regression
equations and correlation coefficients for all
four regions are given in Table 4. There is some
inconsistency in the slopes of these regressions, but we believe that this is due to the
rather narrow range over which the regressions
were calculated and that the least reliable
means (due to rather small numbers of measurements) were obtained for the extremes of
the x axis, thus distorting the slopes slightly.
Nonetheless, the high correlations indicate
that a large percentage of the variance in incremental line distance is explained by the
distance from the DEJ.
Periodicity of incremental lines
The actual time interval (in days) betwen incremental lines was determined, in the labeled
animals. Incremental line distances were divided by the apposition rate for that location,
which had been calculated by the tetyracycline
band measurements as listed in Table 1. The
results established the time, in days, needed to
form the space between two incremental lines.
This time period, frequently described as a
growth rhythm, varied considerably with a
range of 4.19 to 5.83 days (see Table 5). There
was no significant correlation between this
rhythm and distance from the DEJ (r = 0.38,
2
3
4
5
6
7
8
9
10, 1 1 . 12
13
A2
AB
5.11 days
5.83 days
5.05 days
4.68 days
4.63 days
4.47 days
4.83 days
5.09 days
4.45 days
5.06 days
4.50 days
4.68 days
4.86 days
4.47 days
4.71 days
4.59 days
4.37 days
4.58 days
4.19 days
5.32 days
5.22 days
-
'Growth rhythm was calculated by dividing the average incremental
line distance in a tetracycline interval by the apposition rate
for that interval.
P > 0.05), and we see no apparent pattern in
the calculated values of the rhythm.
No change in the incremental line distances
was seen during the intervals 2 and 8, which
were the intervals exhibiting depressed apposition rates, as determined by the tetracycline
measurements. This lack of deviation from expected incremental line distance produced
calculated growth rhythms that were greater
because of the division of distance by a lower
apposition rate figure (see Table 5). Further,
there is no concordancebetween rhythms in the
different areas.
DISCUSSION AND CONCLUSIONS
Our results provided strong evidence that
the pattern of dentinogenesis is a complex process that cannot be reduced to simple characterizations such as the often cited 4 pmlday apposition rate and 4-day 16-pm calcification
rhythm. Over the course of the study, which
spans the middle one-third of dentin development, we were able to document a steady increase in the rate of dentin apposition from 2.6
to 3.9 pmlday in the cervical portion of the
crown. In the intercuspal area a similar increase was noted, and rates were approximately 1 pmlday higher than in the cervical region.
Because our study does not span the entire
growth period, we cannot make definite statements about the rate of apposition during the
entire sequence. However, bearing in mind the
452
S. MOLNAR ET AL.
results of Kawasaki et al. ('77),it is reasonable
to expect that the entire growth of the dentin
probably conforms to the common s-shaped
growth curve and that we are sampling from
the middle of it. Additionally, regional differences in apposition rates are apparent.
There are two factors that can explain these
regional differences. One is differential cell activity; the second is the unique geometry of the
dentin. We have argued elsewhere (Przybecket
al., '79)that an understanding of the cellular dynamics of dentin formation must include a consideration of the increased crowding of the
odontoblasts as they migrate toward the final
contour of the pulp chamber. Indeed, for the
span of our study we were able to document a
declining rate of cell activity in spite of the increasing linear apposition rates. This leads to
the conjecture that geometric considerations
may play a paramount role in the tempo of dentin formation. The phenomenon of cell crowding among odontoblasts as they migrate toward the pulp chamber may cause a simple
reduction in efficiency of the cells by diminution of surface area. Thus the uptake of
materials and production of the dentinal
matrix may be progressively reduced. This
purely mechanical scenario has as its chief advantage the fact that it does not require consideration of complex hormonal processes or
other factors which would regulate odontoblast productivity. In contrast, a nonmechanical model would have to explain both
the change in rate of apposition in any one
region of the tooth and the variation in rate in
different regions of the tooth. The latter aspect,
in particular, would be difficult to model since
we would have to posit differential sensitivity
to controlling factors within a single population of cells.
A mechanistic interpretation of dentinogenesis does not imply that the developing tooth is
immune to metabolic fluctuation. Our data
show that, at least in some instances, agents of
metabolic stress (heat load and cortisone) do
cause depressions in apposition rates. However, where we noted growth depressions, they
were succeeded by periods of compensating acceleration. In the cases in which no growth
depressions were recorded, either the stress
was of insufficient intensity to disrupt normal
dentinogenesis (in the case of the short-term
heat stress), or the tissue markers were administered so as to bracket both the period of
growth depression and the episode of catch-up
growth. (Thisprobably is the case for one of the
cortisone stresses and possibly so for one of the
longer heat stresses.) The response to stress
agents indicates that the forming dentin has
resilience which gives it a certain immunity
from long-term effects resulting from a series
of short-term growth disruptions. The limit of
this resilience clearly was not reached in this
study. Further evidence of this is provided by
the lack of any structural abnormalities in any
of the teeth.
The stress response data raise severalproblematic issues, all of which indicate that conclusions regarding the causes of imperfections
of the dentin may be premature. The intensity,
duration, and mechanism of metabolic stresses
and individual constitution are all probably ingredients in the production of histological
defects in teeth. However, further study must
be undertaken to determine how these factors
interact. We cannot distinguish the contribution of each in our data. For example, would cortisone produce permanent effects if administered for longer periods or in larger dose? Given
these difficulties, it is thus obvious that the interpretation of the presence of microstructural
defects in dentin requires extreme care, contingent on a more precise definition of the etiology
of those defects.
The incremental lines of von Ebner appear to
behave independently of the metabolic fluctuations we observed. During both periods of
depressed growth and accelerated growth, we
were not able to notice any quantitative or
qualitative abnormalities in the spacing of the
alternating light and dark bands of the incremental lines. This indicates that the incremental lines represent structural markers of
dentin growth and are not directly affected by
alterations in the physiological environment of
the tooth and also do not represent the growth
tempo of the tooth to any significant degree.
The exact nature of the structural features
which produce incremental lines is not totally
clear. Schmidt and Keil ('71:lOO) suggested
that the alternating light and dark bands
reflect changes in the orientation of collagen
fibers in the organic phase of the dentin. They
point out that incremental lines can be seen in
decalcified as well as undecalcified sections, indicating that the mineral phase of the dentin
is not involved. It should be noted that
Vincentelli ('78) reached similar conclusions
concerning the laminar structure of the secondary osteon in bone. This interpretation is consistent with our data, but here again we must
refer to the unique geometry of the dentin. The
crowding phenomenon of the odontoblasts and
thus the increased density of the dentinal
tubules probably exerts an influence on the
spacing of incremental lines. The fact that
these lines are more widely spaced near the
pulp may be related to either the primary or
DENTIN APPOSITION RATES AS MARKERS O F PRIMATE GROWTH
secondary curvatures of the tubules. In the
former case, we would suggest that changes in
matrix orientation occur regularly and each incremental line set is produced by an identical or
nearly identical volume of matrix. In the latter case the secondary curvatures or apparent
"buckling" of the odontoblast processes may be
sufficient to cause slight changes in collagen
fiber direction, which cause the optical phenomenon of the incremental line. If this is true,
then the increasing density of odontoblasts
nearer the pulp could mechanically inhibit the
secondary curvature of the tubules and thus
cause the wider spacing of the incremental
lines. In either case the differential odontoblast
crowding in the cervical and intercuspal
regions would account for the observed regional differences.
In sum, we determined that dentin is sensitive to metabolic fluctuation. Variations in
apposition rates are detectable by use of an in
vivo marker of the calcification front. The
naturally occurring incremental lines of von
Ebner do not reflect with accuracy the variations in dentin apposition that are revealed by
tetracycline markers and, thus, are not useful
in detecting apposition rates. The lines do not
vary in width, and there are no changes in optical density during those periods of reduced
apposition rates. However, before definite conclusions can be reached regarding the usefulness of incremental lines, more study is required to determine the precise influence of
tubule density and curvature, and a model
should be employed that treats the tissue as a
three-dimensional object. Such a model would
provide the opportunity to assess tubule density (and thus cell density) as well as allowing the
examination of volumetric relationships, which
may be the most accurate way to study the
complex process of dentinogenesis.
ACKNOWLEDGMENTS
We gratefully acknowledge the support and
encouragement of Dr. Steven Horvath, the Institute for Environmental Stress, University
of California, Santa Barbara. We wish to thank
Mr. William Sawyer and Mrs. Sue Easley for
their technical assistance. Finally, we thank
Iva Molnar for clerical assistance and Marcella
Waddell for preparation of the manuscript.
This project was supported, in part, by NSF
grant SOC75-19191 and NIH DE01453.
453
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