J. Anat. (2008) 212, pp319–329 doi: 10.1111/j.1469-7580.2008.00856.

Regional structural characteristics of bovine

Blackwell Publishing Ltd

periodontal ligament samples and their suitability

for biomechanical tests
Dieter D. Bosshardt,1,2 Marzio Bergomi,3 Giovanna Vaglio4 and Anselm Wiskott4
1
Department of Periodontology and Fixed Prosthodontics, School of Dental Medicine, University of Berne, Berne, Switzerland
2
Department of Oral Surgery and Stomatology, School of Dental Medicine, University of Berne, Berne, Switzerland
3
Laboratory of Applied Mechanics and Reliability Analysis, Swiss Federal Institute of Technology, Lausanne, Switzerland
4
Department of Prosthodontics, School of Dental Medicine, University of Geneva, Geneva, Switzerland

Abstract
Mechanical testing of the periodontal ligament requires a practical experimental model. Bovine teeth are
advantageous in terms of size and availability, but information is lacking as to the anatomy and histology of their
periodontium. The aim of this study, therefore, was to characterize the anatomy and histology of the attachment
apparatus in fully erupted bovine mandibular first molars. A total of 13 teeth were processed for the production
of undecalcified ground sections and decalcified semi-thin sections, for NaOH maceration, and for polarized light
microscopy. Histomorphometric measurements relevant to the mechanical behavior of the periodontal ligament
included width, number, size and area fraction of blood vessels and fractal analysis of the two hard–soft tissue
interfaces. The histological and histomorphometric analyses were performed at four different root depths and at
six circumferential locations around the distal and mesial roots. The variety of techniques applied provided a
comprehensive view of the tissue architecture of the bovine periodontal ligament. Marked regional variations were
observed in width, surface geometry of the two bordering hard tissues (cementum and alveolar bone), structural
organization of the principal periodontal ligament connective tissue fibers, size, number and numerical density of
blood vessels in the periodontal ligament. No predictable pattern was observed, except for a statistically significant
increase in the area fraction of blood vessels from apical to coronal. The periodontal ligament width was up to three
times wider in bovine teeth than in human teeth. The fractal analyses were in agreement with the histological
observations showing frequent signs of remodeling activity in the alveolar bone – a finding which may be related
to the magnitude and direction of occlusal forces in ruminants. Although samples from the apical root portion are
not suitable for biomechanical testing, all other levels in the buccal and lingual aspects of the mesial and distal
roots may be considered. The bucco-mesial aspect of the distal root appears to be the most suitable location.
Key words biomechanical testing; bovine; histology; periodontal ligament.

structure bears witness to its function in tooth positioning

Introduction
The periodontal ligament (PDL) is the structure that
interfaces the teeth with their surrounding alveolar bone.
It allows changes in tooth position (e.g. during tooth
eruption or under orthodontic pressure) and also has a
damping function during chewing (Picton, 1989). It is a
thin and highly specialized soft connective tissue located
between the root cementum and the alveolar bone (for
reviews, see Beertsen et al. 1997; McCulloch et al. 2000). Its
Correspondence
Dieter D. Bosshardt, Department of Periodontology and Fixed
Prosthodontics, School of Dental Medicine, University of Berne,
Freiburgstrasse 7, CH-3010 Berne, Switzerland. T: +41-31-6328605;
F: +41-31-6324931; E: dieter.bosshardt@zmk.unibe.ch
Accepted for publication 14 December 2007
and in the control and dissipation of mechanical stresses as
generated during mastication and traumatic impacts. The
PDL also prevents resorption and fusion of the root with
the surrounding alveolar bone (i.e. ankylosis) (for reviews,
see Beertsen et al. 1997; Andersson & Malmgren, 1999). In
effect, the PDL, root cementum and bone should be
viewed as a structural (Berkovitz, 2004) and functional
(Ivanovski et al. 2006; Benatti et al. 2007) unit that allows
both physiological and therapeutic tooth movements.
Prediction of tooth movements in terms of direction and
magnitude is a central issue in dental biomechanics. In
particular, the characterization of the PDL response to
mechanical stimuli is essential in developing realistic
analytical and numerical tools for the modeling of tooth
displacements in orthodontics, periodontics and the
interactions between the teeth and their restorations.

© 2008 The Authors

Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
320 Structural characterization of the bovine PDL, D. D. Bosshardt et al.
Using finite element analyses, a number of equation
systems (i.e. the constitutive laws) have been proposed
to characterize the biomechanical properties of the PDL
(Pietrzak et al. 2002). They were based on data generated
from human (Mandel et al. 1986; Pedersen et al. 1991; Toms
et al. 2002), rat (Kawarizadeh et al. 2003) porcine (Dorow
et al. 2002; Krstin et al. 2002), and bovine tissues (Pini et al.
2002; Sanctuary et al. 2005, 2006; Shibata et al. 2006).
Mechanical testing of the PDL requires a practical
experimental model. Most importantly, the teeth and the
surrounding bone must be large enough to allow their
fractioning into smaller specimens and still allow their
fastening to the grips of the mechanical testing devices.
Bovine teeth are comparatively large in size and readily
available. As such they qualify as a suitable model substrate
for experimentation. Yet a basic knowledge of anatomy
and histology is a prerequisite for selecting appropriate
teeth or sites around the teeth for biomechanical testing.
In this regard, however, there is an almost complete absence
of information available on normal periodontal anatomy
and histology of bovine teeth. To date, only one study has
illustrated some structural aspects of bovine PDL (Berkovitz
et al. 1997). Unfortunately, many of these are not actually
relevant to the biomechanical response of the tissue.
Therefore the aim of the present study was to assess
those anatomical and histological characteristics of the
bovine attachment apparatus that affect the mechanical
behavior of the PDL.
Materials and methods
Overview
Using freshly prepared sections of bovine molars, the
object of the study was to prepare sets of undecalcified
ground and decalcified semi-thin sections of the PDL from
which the following characteristics were determined:
• size, number and area fraction of blood vessels
• periodontal ligament width;
• profile of the hard tissue interface expressed in terms of
fractal dimensions.
The histological and histomorphometric analyses were
performed at four different root depths (termed ‘a’ (most
apical), ‘b’, ‘c’ and ‘d’ (most coronal)) (Fig. 1) and at six different
circumferential locations around the distal and mesial
roots of the mandibular first molar, that is (Fig. 2A,B): distal
root–distal aspect (‘dd’), distal root–buccal aspect (‘db’),
distal root–lingual aspect (‘dl’), mesial root–mesial aspect
(‘mm’), mesial root–buccal aspect (‘mb’), and mesial root–
lingual aspect (‘ml’).
Preparation of block specimens
Ten bovine mandibles (age range 2–4 years) were obtained
from two local slaughterhouses (Vuillamy, Cheseaux-
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
Fig. 1 Schematic drawing illustrating the sectioning of the mandibular
first molar into four horizontal sections (a–d) of about 1 cm thickness.
sur-Lausanne, Switzerland, and Abattoirs de Clarens, Vevey,
Switzerland) immediately following the death of the
animals. The mandibles were placed in an ice cooler and,
within 30 min, sectioning of the mandibles was initiated
in a specially equipped laboratory of the Swiss Federal
Institute of Technology in Lausanne, Switzerland, following
a method described by Sanctuary (2003). After removal of
all remaining soft tissues using a scalpel, the left and right
sides of the mandibles supporting the molars, premolars
and canines were separated. The specimens were then cut
along the long axes of the third premolar and second
molar to obtain a block containing an intact first molar.
The crowns were removed after sectioning the teeth at the
level of the alveolar crest and, last, blocks each containing
a mesial and a distal root were produced. The cuts were
performed using a heavy industrial band saw (Magnum
Bs 0633, Metabo, Nürtingen, Germany) under abundant
irrigation with saline. The blocks containing one root were
then mounted in a vice and further separated into four
horizontal sections (a, b, c, and d) ~1 cm in thickness
(Fig. 1). While one hemi-mandible was prepared, the other
hemi-mandibles were kept in a refrigerator at 5 °C to
minimize tissue degradation. All newly produced horizontal
root sections were immediately placed in room temperature
fixative solution. Rapidly producing 1-cm-thick horizontal
slices yields a more thorough fixation of the tissues than
immersing larger specimens. Further tissue processing was
performed in the histology laboratory of the Department
of Periodontology and Prosthodontics, School of Dental
Medicine, University of Berne, Switzerland.
Production of undecalcified ground sections
Tissue samples from eight molars were fixed in 4% buffered
formalin for 4 days and then processed for the production
© 2008 The Authors
 Structural characterization of the bovine PDL, D. D. Bosshardt et al. 321

Fig. 2 Low power light micrographs showing

(A) the mesial and (B) the distal roots of the first
mandibular molar. At all root levels, the
horizontal cross-section area was larger at the
distal than at the mesial root, the buccal bone
plate was thicker than the lingual, and the bone
in the interradicular septum was highly
trabecular. The six different locations
investigated were distal root–buccal aspect (db),
distal root–distal aspect (dd), distal root–lingual
aspect (dl), mesial root–buccal aspect (mb),
mesial root–mesial aspect (mm), and mesial
root–lingual aspect (ml). The periodontal
ligament (PDL) was rich in blood vessels (C),
and cells and connective tissue fibers (D).
A–C = ground sections; D = thin section.
AB, alveolar bone; C, cementum; D, dentin;
P, pulp.

of undecalcified ground sections. Briefly, the specimens
were rinsed in running tap water, dehydrated in ascending
concentrations of ethanol, and embedded in methylmetha-
crylate. The embedded tissue blocks were then split horizontally
into approximately thirty 200-μm-thick sections per root using
a slow-speed diamond saw (Varicut® VC-50, Leco, Munich,
Germany). For the histological and histometric analyses,
four sections per root were selected, i.e. one section per
root level. Sections that contained enamel (which in bovine
teeth extends apical to the alveolar crest) and those that
were located too far apically were excluded. Thus, for each
root, four sections approximately the same distance from
each another and devoid of artifacts were produced, resulting
in a total of 64 sections. After mounting the sections onto
opaque acrylic glass slabs, they were ground to a final
thickness of about 100 μm, polished (Knuth-Rotor-3, Struers,
Rodovre/Copenhagen, Denmark) and surface stained with
toluidine blue/McNeal (Schenk et al. 1984). The stained
ground sections were observed in a Leica M8 stereolupe
and a Leica Dialux EB (Leica Microsystems, Glattbrugg,
Switzerland). For polarized light microscopy, the ground
sections were mounted onto transparent acrylic glass slabs
before viewing in the stereolupe.
Production of decalcified semi-thin sections
Five molars were used for the production of semi-thin sec-
tions. Following horizontal sectioning of two mandibular
first molars as described above, the tissue slabs were
fixed for 72 h in 1% glutaraldehyde and 1% (para)formal-
dehyde, buffered with 0.08 M sodium cacodylate (pH 7.4).
Following a brief wash in 0.1 M sodium cacodylate buffer
with 5% sucrose, pH 7.4, the tissue samples were exposed
to a decalcifying solution containing 4.13% disodium
ethylenediamine tetraacetic acid (EDTA). Decalcification
was performed at 4 °C room temperature for 3 months
with constant stirring. The solution was changed twice
weekly. Following decalcification, the tissue slabs were cut
with a razor blade into approximately 50 slices per tooth
in a corono-apical and vestibulo-oral direction. Following
extensive washes in wash buffer solution, 10 slices were
processed for tissue maceration with NaOH, a procedure
that removes cells and noncollagenous extracellular
matrix constituents but preserves collagen structure
and position (Kuroiwa et al. 1992; Macchiarelli et al. 2002).
Tissue slices were immersed in 10% NaOH under con-
stant stirring for 4 days at room temperature. The NaOH
solution was changed daily. Following extensive washes in
wash buffer solution, the macerated and non-macerated
slices were dehydrated in increasing concentrations of
ethanol and embedded in LR White resin (Fluka, Buchs,
Switzerland). Semi-thin sections (1 μm thick) were pre-
pared using glass and diamond knives (Diatome, Biel,
Switzerland) on a Reichert Ultracut E microtome (Leica),
stained with toluidine blue, and observed in a Leica Dialux
22 EB microscope.

© 2008 The Authors

Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
322 Structural characterization of the bovine PDL, D. D. Bosshardt et al.
Size, number and area fraction of blood vessels
The histological slides were analyzed in a light microscope
(Olympus AX 70) at a magnification of 10×. For each of the
6 regions (dd, db, dl, mm, mb, ml) and root levels (a–d),
a set of 7–14 pictures (depending on the width and
morphology of the PDL) was taken in digital format
(640 × 512) to cover a 5 × 5 mm zone. Overlapping pictures
of the total PDL surface were produced. Using a commercial
imaging software (PHOTOSHOP, Adobe Systems, San Jose, CA,
USA), the images obtained from each zone were then
assembled to picture the entire circumference of the PDL
(Fig. 3A). Next, all visible blood vessels (no less than 6 pixel
in diameter) were filled with a white background color to
allow their detection in the subsequent automated steps
(Fig. 3B). The resulting images with white-filled blood
vessels were then processed with a custom-made software
(LABVIEW, National Instruments, Oak Ridge, TN, USA). After
the operator had manually declared a PDL area between
the alveolar bone and the root surface as the region of
interest (ROI), using threshold-based routines, the soft-
ware calculated both the surface (in pixels) of the ROI and
the total surface of the blood vessels. With the aid of a
standard gauge, it was determined that 1 mm corre-
sponded to 770.3 pixels in our system. Hence the program
eventually provided the PDL and the blood vessel areas
([px2] [mm2]), the blood vessel area fraction (percent of
blood vessel total surface vs. PDL surface), the number of
blood vessels contained in the ROI and the numerical
density (number of blood vessels/PDL area [mm−2]). Last,
the diameters of all blood vessels were extracted from the
image. Since the cross-sections of the blood vessels did not
appear as a perfect circles but mostly as ellipses (many
blood vessels are not exactly normal in the transverse
section), it was decided to set the diameters to the small
axes of the ellipses.
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
Fig. 3 One example showing consecutive
pictures (A) before and (B) after all visible blood
vessels in the periodontal ligament (PDL) were
manually selected and filled with a white
background color to ease their detection in the
subsequent automated measuring process.
AB, alveolar bone; C, cementum.
A, B = ground sections.
Periodontal ligament width
The PDL width was assessed in four teeth from two animals
for each root levels (a–d) and each regions (dd, db, dl, mm,
mb, ml). Using the graphic software package, within each
ROI, a line was drawn through the periodontal ligament.
The length of the line was then converted to millimeters,
and the PDL total surface calculated during the blood
vessel measurements was divided by the line length to
yield the PDL width.
Boundaries between PDL and bone and between PDL
and cementum
The fractal dimensions of the two boundary tissues were
assessed to quantify the tortuous windings of their paths.
The following expressions were used to obtain the fractal
dimension of the selected specimens (Krappraff, 1986;
Borodich, 1997):
L(ε) = Kε1−D (1)
L(ε) is the length of the line obtained when using the
measuring unit (or scale) ε. K and D are parameters. L(ε) is
experimentally obtained by
L(ε) = εN(ε) (2)
where N(ε) is the number of times that the unit ε fits into
de measured curve.
The real length L of the curve is thus defined as
L = limε→0 L(ε) (3)
It has been empirically determined that the numbers
N(ε) satisfy the law
© 2008 The Authors

Fig. 4 Thin sections without (A, B) and with
(C, D) prior maceration illustrating the insertion
(arrows) of principal periodontal ligament (PDL)
fibers into the alveolar bone (AB) and the
cementum (C). The maceration process has
removed the cells and some extracellular matrix
constituents but not the collagen fibers.
N(ε) = KεD (4)
from which relation (1) can be derived.
The exponent D in equation (1) is referred to as the fractal
dimension of the line. When D ≈ 1 the boundary is smooth,
whereas for larger Ds the tortuousness of the border is increased.
D is experimentally determined by plotting L(ε) vs. ε on
logarithmic x- and y-scales and then measuring the slope
of the line obtained (slope = 1 – D).
In the present study, four different specimens per location
were examined. ε was taken equal to 1, 1/3, 1/9 and 1/27.
These unit gauges were manually fitted to the intricacies
of the bone and cementum boundaries. The number of
times (N) that the gauge fitted the curve was recorded.
Results
Descriptive histology
For all root levels, the distal root of the mandibular first
molar was wider than the mesial root (Fig. 2A,B). Both bone
© 2008 The Authors
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
Structural characterization of the bovine PDL, D. D. Bosshardt et al. 323
thickness and density varied according to site. Irrespective
of the root depth, the buccal and lingual bone of the
alveolar process consisted of thick cortical bone plates,
whereas the interdental bone septum was extremely thin,
sometimes even non-existent. The interradicular bone
septum was trabecular and porous – the porosity increasing
in the apical direction. The PDL contained a variable
number of large blood vessels (Fig. 3A) and was rich in cells
and fibers (Fig. 3B). The principal PDL fibers were anchored
as Sharpey’s fibers inside the alveolar bone (Fig. 4A,C) and
the cementum (Fig. 4B,D). Tissue maceration prior to
embedding in LR White resulted in the removal of the
cells, leaving the collagen bundles intact (Fig. 4C,D). Due
to active or previously active remodeling activity, fiber
insertion into bone was not present at all sites (Fig. 5A,B).
In general, the morphology of the alveolar bone was
indicative of high bone turnover. In contrast, the remodeling
activity in the cementum appeared to be lower than in the
alveolar bone (Fig. 5A,B). Resorption lacunae were hardly
detected along the cementum surface (Fig. 5C). Soft tissue
channels, however, were frequently observed in the
324 Structural characterization of the bovine PDL, D. D. Bosshardt et al.
cementum, particularly in the apical root portion (Fig. 5D).
These soft tissue channels contained blood vessels. The
presence of Howship’s lacunae without (Fig. 5E) and with
(Fig. 5F) new cementum deposition was indicative of some
remodeling activity in the deeper layers of the cementum.
Along the entire root length, the cementum was of a mixed
type, that is, containing lacunae with both cementocytes
and Sharpey’s fibers (Fig. 5B,E,F).
Figure 6 shows representative micrographs illustrating
the six locations investigated. All six revealed large variations
in PDL width, area fraction of blood vessels, number of
blood vessels, amount and orientation of collagen fiber
bundles, and surface contour of both alveolar bone and
cementum. A qualitative assessment of the six locations at
four different root depths showed the following: (1) In
location dl (Fig. 6A), the cementum and bone contours
were rather jagged and undermined. Blood vessels were
large and numerous; many of them were found in
indentations of the mineralized tissues. Some principal
PDL fibers were functionally oriented. (2) In location dd
(Fig. 6B), the cementum and bone contours appeared in
variable configurations. Blood vessels were numerous but,
in comparison with location dl, fewer were functionally
oriented. (3) In location db (Fig. 6C), the cementum contour
was variable and the bone contour was often jagged.
Blood vessels were numerous where the PDL was thin and
less numerous in zones where the PDL was thick. Many of
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
Fig. 5 Ground (A, C–F) and thin (B) sections
illustrating the remodeling activity of the
alveolar bone (AB) and the cementum (C). At
most sites (A, B, D), the surface contour was
smoother in cementum than in bone. The
irregular and jagged surface contour of the
alveolar bone, the marked differences in the
staining intensity between bone matrix
compartments (A), and the presence of
osteoclasts (arrows) were indicative of a high
bone remodeling activity. In contrast, the
cementum surface revealed only sparse
resorption lacunae (C). However, soft tissue
channels (asterisks) were frequently observed in
the apical cementum (D). These soft tissue
channels contained blood vessels (BV). The
presence of both Howship’s lacunae
(arrowheads) and newly deposited cementum
matrix (NC) indicated remodeling activity in the
depth of the cementum layer (E, F). D: dentin;
PDL: periodontal ligament.
the blood vessels were located in hard tissue indentations
and a sizeable amount of the collagen fibers were oriented
in parallel bundles, particularly at wide PDL sites. (4) In
location ml (Fig. 6D), the cementum and bone contours,
the number of blood vessels, and connective tissue fiber
orientation were variable. (5) In location mm (Fig. 6E), the
cementum contour was flat and the bone contour was a
little jagged. The number of blood vessels was variable
and many collagen fibers were functionally oriented. (6) In
location mb (Fig. 6F), the cementum and bone contours
were essentially flat, the number of blood vessels was
variable, and some of the collagen fibers were functionally
oriented.
Histomorphometry
Blood vessel surface
The area fraction of blood vessels is presented in Fig. 7.
The lowest and highest absolute values for the area fraction
of blood vessels were 7.0% and 34.2%, respectively, and
the values pooled per region varied from 11.9% (dl) to
23.5% (dd). ANOVA and Scheffe’s analyses revealed a sig-
nificant (P < 0.05) difference between locations, that is,
the more coronal the section, the larger the area fraction
of blood vessels. The lowest and highest absolute numbers
of blood vessels per mm2 were 26 and 112, respectively,
and the data pooled per region ranged from 28.0 (dl) to
© 2008 The Authors
 Structural characterization of the bovine PDL, D. D. Bosshardt et al. 325

Fig. 6 Ground sections illustrating the six

locations investigated: (A) dl, (B) dd, (C) db,
(D) ml, (E) mm, and (F) mb. Large variations in
periodontal ligament (PDL) width, area fraction
of blood vessels (BV), number and size of blood
vessels, amount and orientation of collagen
fibers, and surface contour of both the alveolar
bone (AB) and the cementum (C) may be seen.

Fig. 7 Percent area fraction of blood vessels

for the four root levels, a (apical) to d (coronal).

49.72 (ml). The blood vessel diameters at the buccal
locations were significantly greater (P < 0.05) than at all
other locations, and the smallest blood vessels were found
at the lingual location of the distal root.
PDL width
The PDL widths are presented in Fig. 8. The mean PDL
width for all 95 measurements was 551 ± 125 μ m
(mean ± SD) with the lowest and highest values meas-
uring 221 μm and 785 μm, respectively. For all locations,
there was an increase of the PDL width from coronal to
apical.
Fractal dimensions of the cementum and bone surfaces
The fractal dimensions of the cementum and bone con-
tours are shown in Table 1. The fractal dimensions of the
cementum and alveolar bone ranged from 1.01 to 1.10
and from 1.06 to 1.22, respectively. For all locations except
‘dd’ the fractal dimensions of the bone contour were
higher than those of the cementum surface.
Polarized light microscopy
Polarized light microscopy was performed to better visualize
the density and architecture of the PDL collagen fiber

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Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
326 Structural characterization of the bovine PDL, D. D. Bosshardt et al.
Table 1 Fractal dimensions (D) for cementum and bone at the six
peripheral locations
Cementum Bone contour
Location contour (#specimens)
dL (4) 1.10 ± 0.05 1.22 ± 0.08
dd (4) 1.09 ± 0.06 1.06 ± 0.03
db (4) 1.08 ± 0.05 1.18 ± 0.05
mL (4) 1.05 ± 0.02 1.07 ± 0.04
mm (2) 1.01 ± 0.02 1.14 ± 0.04
mb (4) 1.01 ± 0.01 1.04 ± 0.01
bundles. The qualitative assessment showed that both
fiber density and orientation varied greatly among sites
(Fig. 9). In general, fibers were more densely packed and
displayed a higher degree of orientation at root levels b,
c, and d (Fig. 9A,B), whereas the apical PDL (i.e. at level a)
was wider and contained fewer and less organized
connective tissue fibers (Fig. 9C,D).
Discussion
The present study is part of a research line aimed at char-
acterizing the mechanical characteristics of PDL (Pietrzak
et al. 2002; Pini et al. 2004; Justiz, 2004; Shibata et al.
2006). The testing principle consists in loading samples
under well defined conditions, that is, by controlling 1)
the origin and structure of the tissue, 2) the geometry of
the specimen, 3) the mechanical preconditioning of the
sample, 4) the load profile applied to the tissue and 5) the
physical environment (moisture, atmospheric pressure).
The data sets thus generated then provide the basis for
equations systems (i.e. the constitutive laws) which simulate
the response of the three compartments controlling the
dynamics of a tooth under load, that is, 1): the fibrous
component (collagen and elastic fibers), 2) the ground
substance and 3) the vasculature. Ideally, such equation
sets should be ‘robust’ in that the equations that model the
behavior of the tissue should be controlled by parameters
that reflect the structure and properties of the tissue (and
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
Fig. 8 Periodontal ligament width for the four
root levels, a (apical) to d (coronal).
should not be written as any ‘best fit’ function). Further,
for a given tissue type, the basic response should be modeled
within the equation system. Refinements pertaining to
species should be obtained by adjusting tissue-specific
parameters.
The drawback of this approach is that it discounts the
stabilizing effect of the vasculature and does not (as yet)
establish a link between local tissue response and global
tooth movements. Both these issues have been discussed
in previous reports (Pini et al. 2002; Sanctuary et al. 2005;
Sanctuary et al. 2006).
Bovine vs. human teeth
The PDL is part of a multicomponent unit forming the
tooth attachment apparatus. Therefore, a functional
characterization of the PDL cannot be confined to the
soft tissue but must take the two bordering mineralized
tissues, that is, the alveolar bone and the cementum, into
consideration. For the first time, the present study
characterized the PDL of the bovine mandibular first
molar both morphologically and morphometrically. The
various histological techniques used provided a com-
prehensive insight into the complex structural organization
of the tooth attachment apparatus in bovine mandibular
first molars and demonstrated marked species-specific
particulars and differences with human tissues.
The number of fibers, their thickness and density,
structural organization and insertion modus into both
alveolar bone and cementum are all parameters that may
affect the attachment function. At a number of sites, the
periodontium of bovine teeth shows histological signs of
active or post-active remodeling activity and suggests that
the local attachment function is weakened at such sites.
The higher fractal dimensions of the bone surface compared
with the cementum support the qualitative observations
of a higher remodeling activity in the alveolar bone, since
an increase in the number of resorption lacunae results in
a more irregular surface profile. If the remodeling activity
is confined to tiny spots, the overall attachment function
may not be affected. However, no predictable distribution
© 2008 The Authors
 Structural characterization of the bovine PDL, D. D. Bosshardt et al. 327

Fig. 9 Ground sections viewed under

polarized light showing non-uniform fiber
density and orientation. The periodontal
ligament (PDL) fiber system connecting the
cementum (C) with the surrounding alveolar
bone (AB) was either straight (A), angulated
(B), or arranged in a criss-cross pattern (C, D).
In contrast to the root levels b–d (A, B), the
apical periodontal ligament presented a low
fiber density and a lower degree of fiber
organization (C, D). D, dentin.

pattern of resorptive activity could be deduced from the
present material. When compared to bovines, human
teeth have a lower incidence of remodeling activity. This
observation is probably related to different mastication
patterns as cattle are ruminant herbivores with extreme
horizontal tooth displacements during grinding. Although
most remodeling activity was observed in the alveolar
bone, the root cementum also showed histological signs of
moderate remodeling activity, albeit to a much lesser
degree than in the alveolar bone. The permanent eruption
of bovine molars results in the formation of thick layers of
cementum on the roots and the cervical portion of the
crown (Ainamo, 1970). This continuous process, which
requires rapid cementum deposition, may contribute to
the high remodeling activity in the bovine periodontium.
An interesting finding was that the cementum, particularly
in the apical root portion, contained soft tissue channels
with blood vessels. This is in contrast to human teeth which
are not known to present vascularized soft tissue channels
inside the cementum (for a review, see Bosshardt, 2005). It
suggests that cementum in the apical root portion has
more bone-like features in bovine than in human teeth.
The presence of blood vessels may enable more cementocytes
to survive in the deeper layers and/or to play a role in
cementum remodeling.
Site-specific characteristics of the bovine attachment
apparatus
In contrast to the surface-stained ground sections viewed
under bright field illumination, polarized light microscopy
allowed a more detailed visualization of the PDL connective

© 2008 The Authors

Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
328 Structural characterization of the bovine PDL, D. D. Bosshardt et al.
tissue fibers. These images demonstrated a high degree of
randomness in terms of fiber density and orientation ranging
from compact and highly organized architectures to
rather low fiber density and organization. In the most
apical root segment, fiber density was lowest and its
architecture showed the least degree of structuring.
There was one site, however, which quite consistently
demonstrated a high degree of fiber organization. This
site was located at the bucco-mesial aspect of the distal
root at levels b, c and d. Since the fibers are predictably
organized and the buccal bone plate is very thick, these
sites may be regarded as the most suitable locations for
harvesting samples for mechanical testing.
A remarkable difference between the bovine mandibular
first molar and human teeth is the distribution pattern of
types of cementum. While the coronal half of human roots
is covered with acellular extrinsic fiber cementum, and
the apical root portion and the furcation is covered with
cellular mixed stratified cementum, there was only one
type of cementum found in bovine teeth, that is, a mixed
type consisting of embedded cells (i.e. cementocytes) and
Sharpey’s fibers. This has implications on stress distribution
and dissipation around the roots as, due to its high
number of inserting Sharpey’s fibers, acellular extrinsic
fiber cementum is a major contributor to the attachment
function (Bosshardt & Selvig, 1997). In general, in the
bovine molar, there appears to be a lower but more homo-
geneous number of fibers inserting into the cementum
along the root.
The maceration of the tissue samples removed cells
and parts of the noncollagenous proteins but leaving the
collagen structure intact. Thus far, the NaOH maceration
has been used to visualize fiber architecture in scanning
electron microscopy. To our knowledge, this is the first
time that maceration was used in combination with tissue
embedding, sectioning and optical microscopy. This
methodology may be applied in future studies to assist in
calculating the collagen fiber area fraction in soft connective
tissues using computer-aided image processing.
The measurements of size, number and area fraction of
blood vessels showed values that varied greatly from one
location to another. The higher area fraction of blood
vessels in the coronal PDL portion may be explained by
a contribution of branches of the interalveolar and
interradicular arteries, which extend coronally inside the
spongiosa of the alveolar process (Schroeder, 1986).
Like the blood vessel measurements, large variations
were noted in PDL width. All measurements, however,
were clearly superior to the PDL width of human teeth,
which is in the order of 100–300 μm (Coolidge, 1937;
Schroeder, 1986).
The highest values in the bovine thus are two to three
times higher than in humans. It is known that human teeth
respond to increased functional loads by a widening of their
PDL space (Schroeder, 1986). Therefore the greater PDL
Journal compilation © 2008 Anatomical Society of Great Britain and Ireland
width of the bovine teeth may have been caused by higher
functional loads in herbivores. In human teeth, the PDL is
always thinnest at the mid-level of the root (Schroeder,
1986) whereas in the present study a consistent increase in
PDL width from coronal to apical was noted. This observation
implies that samples for biochemical testing should be
extracted from similar root levels (although large differences
were observed between animals as well).
Conclusions
The present study provided the baseline information on
the tooth attachment apparatus in bovine mandibular first
molars which should assist in planning and conducting
future biomechanical studies of the PDL. The histological
observations and the histomorphometric data indicate
that the morphology of the PDL is highly inhomogeneous
around a given tooth and between teeth without pre-
dictable pattern between sites and animals. While the
most apical root portion is not recommended for harvesting
samples for biomechanical tests, the buccal and lingual
aspects of the mesial and distal root – along with the bucco-
mesial aspect of the distal root – appear to be the most
suitable locations.
Acknowledgments
The authors gratefully acknowledge the technical assistance of
Mrs M. Aeberhard and Mrs E. Wagner and the help of Professors
J. Botsis and U. Belser. This study was supported by the Swiss
National Science Foundation (grants no. 3252-068162.02/1 and
21-64562.01).
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