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Three-dimensional reconstruction of the rat incisor by means of computerized histomorphometry.

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THE ANATOMICAL RECORD 205:455-464 (1983)
Three-Dimensional Reconstruction of the Rat Incisor by Means of
Computerized Histomorphometry
Department of Orthodontics (S.S.),Anatomy and Embryology WM., M. WJ,
and Expeririiental Medicine and Cancer Research (G.Z.), Hebrew
Uniuersity-Hadassah Dental and Medical Schools, Jerusalem, Israel
A detailed, quantitative, three-dimensional reconstruction of
the adult rat lower incisor is presented. The mandibles of 12 male adult rats
were dissected and radiographed. The outline of each incisor was traced and
the center of the arc formed by the lower, or labial, border of the tooth was
geometrically determined. The sector of the bone-embedded part (mean length
20.70 _+ 3.5 mm) of the tooth was divided into six equal segments by radii
drawn through the arc center. The dividing radii were transposed onto the
specimens and three consecutive 100-pm transverse ground sections were cut
from the incisal side of each segment, parallel to their bordering radii. The
distance between each section and posterior wall of the alveolar socket was
measured and the information stored in the computer. The outlines of all the
dental structures were traced and fed into a computer with the aid of a sonic
digitizer. The circumference of the tooth (5.91
0.0021, and the mesiolateral and labiolingual widths (1.36 + 0.001 mm and 2.18 + 0.01 mm,
respectively) remained constant at all tooth levels. The decrease in width and
cross-sectional area of the pulp and the corresponding increase in dentin were
represented by the best fitted second order polynomials. These changes were
not uniform, being more pronounced in the proximal part of the tooth. The
polynomials also served for calculation of the total volumes of pulp, dentin,
and enamel (13.93 mm3, 18.05 mm3, and 4.43 mm3, respectively). The threedimensional measurements included the periodontal ligament (PDL), which
was significantly wider on the lateral side than on the mesial (0.16 & 0.006
mm and 0.12 & 0.004 mm, respectively). The volumes of the enamel-bordering
PDL and of the cementum-bordering PDL were 7.77 mm3 and 8.67 mm3,
respectively. In the latter, the tooth-related compartment constituted 42.7% of
the total volume. The data presented can serve as a basis for future quantitative studies of the rat incisor.
The constant process of tissue renewal in
the rat incisor provides a n excellent model
for a study of a variety of fundamental biological problems. Consequently, a profusion
of investigations on tooth morphology, with
particular stress on the proliferative basal
and odontogenic organ, have been and are
being carried out. The early works were restricted to two-dimensional studies of tooth
morphology, the most detailed one being that
of Addison and Appleton (1915). In the 1930s,
Schour and Steadman (1935) attempted a
three-dimensional generalized description of
the rat incisor in connection with the pattern
of apposition of dentin and enamel. Later,
0 1983 ALAN R. LISS, INC.
more sophisticated approaches employing
both longitudinal and cross-sectional planes
of reference were applied to the study of the
various tooth structures (Pindborg and Weinman, 1959; Chiba, 1965; Robins, 1967; Lavelle, 1968; Moe, 1971; Michaeli and Greulich,
1972; Warshawsky and Smith, 1974; Smith,
1975; Smith and Warshawsky, 1975). In these
and other investigations of rat incisor morphology, upper and lower as well as left and
right incisors were correlated (Smith and
Warshawsky, 1973; Smith, 19751, age
Received October 27,1982; accepted January 12, 1983.
changes explored (Lavelle, 19681, and the incisor compared to the teeth of primates (Warshawsky et al., 1981).
The three-dimensional representations of
the tooth either lack quantitative data
(Smith, 1975; Smith and Warshawsky, 1975,
1976) or concentrate upon the odontogenic
organ only (Michaeli and Greulich, 1972).
Therefore, in spite of a n abundance of investigations, the basic quantitative data on the
various tooth components are still missing.
The purpose of the present study is to obtain,
by means of a specially designed, computerized image analysis technique, detailed threedimensional quantitative information about
the adult rat incisor as a whole and, at the
same time, gain data about each of its separate constituent tissues at any given level
along the tooth.
Fig. 1. Lateral radiograph of a dissected rat mandible.
Twelve male adult rats of the Sabra strain
(mean weight 230 k 10 g) were used in this
experimental work. The mandibles were removed, disssected free of soft tissue, halved
through their midline and fixed in BouinHolland fluid. Orthoradial nondistorted lateral radiographs were taken of each mandible (Fig. 1). The fixed mandibular incisors
were then divided into six equal segments by
a method which eliminates the distortion
caused by the tortuous shape of this tooth
(Fig. 2). Namely, each radiographed mandibular bone and its tooth were traced on an
acetate sheet, and the center of the arc
formed by the labial border of the boneembedded part of the incisor was geometrically determined. From this center, two radii
were drawn-one to the posterior limit of the
alveolar socket, and the second to the ante-
b-pdl, bone-related
Lin, lingual
pdl, periodontal
t-pdl, tooth-related
blood vessel
Fig. 2. Schematic drawing showing the division of the
rat incisor into six (a to D equal segments.
rior limit of the lingual alveolar bone. The
arc of the segment bordered by these two
outer radii was measured, and six equal parts
were marked off. Radii passing through these
points of measurements were drawn, dividing this tooth section into segments of identical size.
The left mandibles were mounted on an
Isomet low-speed saw (Buhler Ltd.), and three
consecutive 100-pmthick undecalcified transverse sections were cut from the incisal side
of each of the six segements, parallel to their
bordering radii (Fig. 3). The micrometer,
which forms a part of the Isomet saw, accurately recorded the thickness of each cut section of the tooth. The length of the remaining
sector was measured precisely using a fine
caliper connected to a digital voltmeter.
These measurements established the exact
distance of each ground section from the posterior limit of the alveolar socket.
Fig. 3. Cut cross sections of rat incisor from segments a, b, c, d (see Fig. 2).
The sections were viewed on a Reichert
Visopane microscope, and the outlines of the
tooth components and its surrounding bone
were traced. The tracings were fed into a
computer with the aid of a sonic digitizer.
Each curve was double-traced with a special
stylus, and the coordinates of each point were
introduced into a PDP-15/20 computer and
displayed as digitized tracings on a storage
scope for visual inspection. Special programs
computed the dimensions of the different anatomical structures.
The following cross-sectional parameters
were measured (Fig. 4):
1. Total tooth dimensions: circumference
with and without the enamel layer; mesiolatera1 width (as expressed by line 1-1' joining
the mesial and lateral cementoenamel junctions); labiolingual width (as expressed by
line 2-2'). To establish the latter, a point on
the lingual tooth outline (2) representing the
maximal distance between this outline and
the midpoint on the cementoenamel junction
plane was determined. The maximal distance between point (2) and the labial outer
surface (point 2') was then measured.
2. Pulp: circumference; mesiolateral width
along the cementoenamel junction plane;
maximal labiolingual width.
3. The width of the dentin measured between its outer and inner (including predentin) boundaries along lines drawn in the
Fig. 4. Tracing showing the location of lines used in
the measurements. For detailed explanation, see text.
center of the labial, lingual, mesial, and lateral dentinal walls, perpendicular to the
outer surface (line 3); width of the enamel
(line 4)as measured along the line of maximal tooth width.
4. The periodontal ligament (PDL) was divided by the cementoenamel junction plane
into its two anatomically different compartments-i.e., the enamel- and the wmentumbordering ligament, respectively. The width
of the former was measured along the median perpendicular to the cementoenamel
junction plane. To calculate the mesial and
lateral periodontal width, the cementum-bordering PDL was arbitrarily subdivided into
mesial, lateral, and lingual sections by a n
arc tangent to the outer lingual tooth outline
and having as its center the midpoint of the
cemento-enamel junction plane. The division
of the areas of the mesial and lateral sections
by their measured length yielded the periodontal space width.
The cementum-bordering PDL was subdivided into its two morphologically different
compartments (Fig. 5A)-i.e., the tooth- and
the bone-related components (Beertsen,
1973). In order to measure the dimensions of
each of these compartments separately, the
lower right mandibles were decalcified in
10% EDTA, divided into six segments, using
the same technique as for the left incisor,
and embedded in paraplast. Sections (6 pm)
were cut from the incisal side of each of the
six segments, parallel to their bordering radii, and stained with hematoxylin-eosin. The
sections were subjected to histomorphometric procedures as described above and, in
addition, a demarcation line between the two
compartments was drawn (Fig. 5B), enabling
their separate evaluation. The most proximal sections of the tooth, which are not completely surrounded by bone, were excluded.
The measurements obtained from all
ground sections were plotted according to
their distance from the posterior wall of the
alveolar socket (abscissa), and second-order
polynomials were fitted to these points (Michaeli et al., 1981). In these polynomials the
value y depicts the tissue parameter dependent upon the distance of the tissue (x) from
the tooth origin. The adequacy of the polynomial fit was checked using the x2 test. The
polynomials did not deviate significantly
from the observed points, thus rendering
them valid for the derived computations. The
cross-sectional areas of pulp, dentin, and enamel and their total volumes, as well as the
volumes of the different parts of PDL, were
Fig. 5. The division of the periodontal ligament into
tooth-related and bone-related compartments. A) Histo
logical section showing the clearly defined demarcation
line (black arrows); B) tracing. Hematoxylin-eosin. X750.
computed by specially designed programs.
Student’s t-test was used for statistical evaluation of the results.
In the present investigation the measured
and calculated values were obtained from
that bone-embedded part of the rat incisor
where dentin secretion already fully surrounds the pulp-that is, approximately 4
mm from the posterior alveolar wall. From
this point on, the tooth was perfectly cylindrical, presenting identical dimensions of
cross-sectional widths and dentin circumference along the entire length (Fig. 6B, C, D).
Proximally (9 mm), the total tooth circumference showed a very slight increase, reflecting
the process of enamel apposition (Fig. 6A).
The tooth dimensions are given in Table 1.
Since the dimensions of the pulp reflected
the rate of dentinogenesis, these two tooth
components will be described together. The
cross-sectional dentin area continuously increased, reaching 2 mm2 a t the alveolar border, whereas the corresponding pulp area decreased from 2 mm2 to 0.1 mm2 (Fig. 7A, B).
These dimensional changes did not proceed
uniformly, being more pronounced proximally (up to 13 mm), with considerable deceleration toward the alveolar crest.
The augmentation in the width of the dentinal walls was identical on the mesial, lateral, and labial aspects of the tooth, the last
aspect remaining thicker throughout by approximately 50 pm. As already reflected by
the areal dimensions, there was a n initial
steep increase in dentinal width along the
apical half of the tooth, slowing down distally
(Fig. 8B). The lingual dentinal wall (Fig. 8A)
showed a different picture, as expressed by
the polynomial fitted to its measurements.
The steep increase in thickness proceeded
steadily toward the distal tooth end, culminating in a mean dentinal width of 0.74 mm
at the level of the alveolar crest. This was
considerably thicker than the mesial dentin
(0.51 mm) at the same level (P < 0.001).
Consequently, the labiolingual width of the
pulp decreased almost uniformly from a
mean of 1.9 mm a t the apical end to 0.7 mm
at the distal aspect, whereas the decrease in
the mesiolateral width (from 1.2 mm to 0.1
Fig. 6. The overall tooth dimensions. A) Tooth circumference; B) outer dentin circumference; C) cross-sectional
mesiolateral width; D) cross-sectional labiolingual
TABLE 1. The measured and calculated parameters of dental tissues in adult rat incisors
Total parameter
Bone-embedded length
Mesiolateral width
Labiolingual width
Dentin: circumference
Width (distal 5 mm)
Cross-sectional area (distal 5 mm)
Periodontal ligament
Cementum-bordering(tooth-related part)
Cementum-bordering (bone-related part)
Total mesial width
Total lateral width
Tooth-related mesial width
Tooth-related lateral width
Tooth-related lingual width
mm) occurred mainly along the proximal 13mm part of the tooth length (Fig. 9). Therefore, the contour of the pulp constantly
changed, the ratio of its mesiolateral to labiolingual dimensions diminishing from 0.64
a t the apical end to 0.14 a t the alveolar crest
level. The cross-sectional enamel area and
Mean size (mm)
Total volume (mm3)
f 0.350
f 0.020
f 0.010
f 0.010
5.53 f 0.030
0.14 f 0.003
0.28 f 0.005
0.12 f 0.004
0.16 f 0.006
0.052 ? 0.003
0.057 f 0.003
0.105 f 0.006
width continued to increase in the proximal
8 mm of the tooth, after which these parameters remained constant (Fig. 10).
The enamel-bordering PDL, which in the
rat incisor does not contain tooth anchoring
collagenous fibers, exhibited a regular outer
border. It was widest a t the proximal tooth
end (0.43 mm), gradually lessening to a constant mean value of 0.18 mm in the distal
half of the tooth (Fig. 11).
In the cementum-bordering PDL, the outer
border and, as a result, the cross-sectional
PDL area, varied in the different section.
This was particularly evident on the lingual
tooth aspect. Therefore, only the total PDL
volume and the mean width of the mesial
and lateral PDL were estimated (Table 1).
The lateral PDL was significantly wider than
the mesial one (P < 0.001).
The tooth-related PDL constituted 42.7% of
the total PDL volume (Table 1).It had fairly
regular outlines, with the mesial and lateral
sections having a n almost similar width, and
the lingual aspect being nearly double their
thickness (Table 1; Fig. 12).On all these three
sides, the width of the tooth-related PDL, as
well as its area (Fig. 13B), increased in the
proximal part of the tooth, reaching constant
values at about 8-10 mm from the posterior
wall of the alveolar socket. The measurements of the bone-related PDL area (Fig. 13A)
yielded widely divergent values with a tendency to similarity along the tooth length.
Fig. 7. Graphic presentation and polynomial equation
of the cross-sectional area of pulp (A) and dentin (B). y~
= 2.92 - 0 . 2 5 ~
0 . 0 0 6 ~ YB
~ ; = -0.74 + 0 . 2 3 ~-
The few three-dimensional reconstructions
of the basal part of the rat incisor described
in the literature (Michaeli and Greulich,
1972; Smith and Warshawsky, 1975) were
executed by the painstaking method of gluing
together thin plates of material representing
enlarged cross sections of the tooth. The application of computerized image analysis facilitated the development of a new and
simpler method for the three-dimensional
demonstration of the tooth as a whole, and of
each of its tissues separately.
The lower rat incisor is difficult to reproduce, as it is both curved anteroposteriorly
and twisted mesiolaterally, comparable to "a
portion of a flattened spiral" (Addison and
0.6 -
Fig. 8. Graphic presentation and polynomial equation of the cross-sectional width of the lingual (A) and
mesial (B) dentin. y~ = 0.22 + 0 . 0 5 ~- 0.0001~~;
YB =
-0.18 + 0 . 0 5 ~- 0 . 0 0 0 6 ~ ~ .
Fig. 9. Graphic presentation and polynomial equation of the pulp circumference (A) and cross-sectional
labiolingual (B) and mesiolateral C) width. y~ = 6.73 -
2'0 mrn
Fig. 10. Graphic presentation of the cross-sectional
area of enamel and polynomial equation for the proximal
8 mm of this tissue. y = -0.09 + 0 . 0 6 ~- 0 . 0 0 1 4 ~ ~ .
2 0 mm
Fig. 11. Graphic presentation and polynominal equation of the cross-sectional width of the enamel-bordering
periodontal ligament. y = 0.53 - 0 . 0 5 ~+ 0 . 0 0 1 7 ~ ~ .
0 . 3 1 ~+ 0 . 0 0 3 ~YB
~ ; = 2.40
1.85 - 0.13~+ 0 . 0 0 2 ~ ~ .
0 . 0 9 ~- 0 . 0 0 0 4 ~yc
Appleton, 1915).We have endeavored to overcome this difficulty by dividing the tooth into
relatively small, straight segments and sampling each of these segments perpendicularly
to its own axis of length. The degree of accuracy obtained by our method is reflected in
the measurements of the total tooth dimensions. As the continuous apposition of dentin
progresses inward from the external perimeter of the tooth, the previously established
circumference at the proximal tooth end has
to retain its shape and size. And, indeed, the
outline and width of the teeth measured in
our study produced identical values at all
levels of sectioning, offering proof that no
element of distortion was present.
The new method described here enables
the measurement and calculations of the parameters of various dental tissues. The
amounts of dentinal and pulpal tissues
change incessantly from the proximal to the
distal tooth extremities, prohibiting calculation of total mean values for these tissues.
Thus, each parameter has to be evaluated
separately at any given level, using the fitted
polynomials. The continuous addition of new
dentin and the consequent narrowing of pulp
space did not proceed uniformly. To understand this phenomenon, one has to view the
distance of any given section of tissue as a
function of time. The dentin-secreting cells,
maturing near the tooth origin, are passively
carried by the tooth eruption toward the distal tooth end, where they die and are eliminated by the process of attrition. As the daily
of the values obtained for dentin shows that,
starting from approximately the age of 29
days (13 mm distant from the proximal end),
the rate of apposition by the older cells is
slowed down. This might be explained by
either the process of aging of the secreting
cells or the environmental changes of the
progressive pulp obliteration. An interesting
finding was the exceptional behavior of the
lingual dentin, which not only failed to show
deceleration in the process of its increasing
width, but even exhibited a certain accelera0.21
tion. This is a n example of a response to
physiological demands upon tissue, as the
lingual dentin at the distal tooth end directly
bears the impact of the attritional activities
of the animal.
I " " l " " I " " l " " I ~
20 m m
The data obtained on enamel width tally
with those already published in the literaFig. 12. Graphic presentation and polynomial equature (Addison and Appleton, 1915;Schour and
tion for the proximal 8 mm of the cross-sectional width
of the lingual (A),mesial (B), and lateral (C) tooth-related
Steadman, 1935). However, it has to be
periodontal ligament. y~ = 0.13 + 0 . 0 5 ~- 0 . 0 0 3 ~yB
pointed out that they represent only the
= -0.13 + 0 . 0 5 ~- 0 . 0 0 4 ~ yc
~ ; = 0.14 - 0 . 0 4 ~+
maximal width of the labial surface while
the enamel layer tapers toward the mesial
and lateral cementoenamel junction. Warshawsky and Smith (1974) have found that
the length of the presecretory and secretory
zones of an enamel organ equals 7.2 mm. In
keeping with their findings, our results, too,
show a secretory activity approximating8 mm
in length, as measured from the posterior
wall of the alveolar socket; because of its constantly increasing width, the area of enamel in this part of the tooth has to be expressed
by its polynomial.
In the present work, the detailed quantitative investigation of the PDL is an additional
feature in the accumulating knowledge about
the rat incisor. The increment in the dimeno.31
sions of the periodontal space on the lateral
and lingual sides accounts for the increased
tooth mobility in these two directions. It is
interesting to note that, in spite of the difference in the total width of the mesial and
lateral PDL, the tooth-related PDL on these
sides was nearly equal. The increment in the
width of the lateral PDL has therefore to be
ascribed to the bone-related, blood vesselFig. 13. Graphic presentation of the cross-sectional
containing ligament. It has to be rememarea of the bone-related (A) and tooth-related (B) cemenbered that the findings on the PDL in the
tum-bordering periodontal ligament and polynomial
equations for A and for the proximal 8 mm of B. y~ =
present work were obtained on healthy, nor0.24 + 0.02X - o.ooo7X2;yB = -0.13 + 0 . 0 7 ~- 0 . 0 0 4 ~ ~ . mally erupting incisors. Whether these values for bone- and tooth-related PDL are
applicable under changed functional derate of eruption of the rat incisor is constant,
mands is now under investigation.
amounting to 450 pmlday, the division of the
The data presented in this study may serve
distance of a given section from its basal end
as a baseline for future quantitative experiby this value establishes the age of the odonments on the rat incisor. One of the main
toblasts in this specific section. Examination
merits of the described method is the ability
to calculate tissue volumes. As such, it can
also be utilized to measure precisely the volumes of fragments of dentin or enamel used
for chemical analysis, or to establish tissue
volume in any given segment of the tooth.
The three-dimensional reconstruction of the
dental cell environment, together with the
knowledge of daily celI movement (derived
from measuring tooth eruption), can provide
kinetic information without resorting to the
use of isotopes and autoradiography. For example, this method was successfully applied
to the study of kinetics of the odontoblast
population of the rat incisor (Weinreb et al.,
1982) and the rate of dentinogenesis (Michaeli et al., 1982).
The computerized histomorphometric method, which in our study was applied to the
detailed portrayal of the rat lower incisor
(Fig. 14), can be successfully used for the
Fig. 14. Example of a computerized three-dimensional reconstruction of lower rat incisor obtained from
cross-sectional tracings at various levels along the tooth.
quantitative description of any anatomical
structure, provided it has a clearly defined
shape. If the replicating cells in the structure
present a steady state, the method can also
be applied to kinetic investigations.
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