Periodontology 2000. Vol.
13, 1997, 20-40
Printed in Denmark. All rights reserved
The periodontal ligament:
a unique, multifunctional
connective tissue
BEERTSEN,
WOUTER CHRISTOPHER
A. G. MCCULLOCH
& JAROSLAV SODEK
The periodontal ligament is the soft connective
tissue interposed between the roots of our teeth and
the inner wall of the alveolar socket. Its fibers form a
meshwork that stretches out between the cementum
and the bone and is firmly anchored by Sharpey’s
fibers (Fig. 1). The periodontal ligament links the
teeth to the alveolar bone proper, providing support,
protection and provision of sensory input to the
masticatory system. The fibroblasts of the ligament
originate in part from the ectomesenchyme of the
investing layer of the dental papilla (181, 1831, and
this developmental origin may give these cells spe-
cialized properties. In a number of respects they are
different from cells in other connective tissues. Their
properties are addressed in this chapter, both from
a morphological and a functional point of view. Our
objective is to illustrate how the unique properties
of the periodontal ligament endow this tissue with
functional attributes that are not replicated by other
tissues. Where possible, reference will be made to
studies on the periodontal ligament of human teeth.
The reader should always keep in mind, however,
that very little information about the periodontal
ligament has come from direct observations in
humans.
How unique is the periodontal
ligament?
Experiments in dogs (92), monkeys (4, 127) and ro-
dents (201) have shown that when periodontal liga-
ment cells are removed from the cementum by
mechanical means or displaced or altered under the
influence of certain compounds, such as the bispho-
sphonate 1-hydroxyethylidene-1,l-bisphosphonate,
ankylosis may occur. Bone tissue invades the peri-
20
Coavricrht 0 Munkscraard 1997
PER
10DoNTO LO GY 2 000
ISSN 0906-6713
odontal ligament space and establishes a direct con-
nection between the tooth and the wall of the al-
veolar socket. Although such ankylosis can exist for
some time, this nonresilient type of tooth support
usually leads to loss of function and to resorption of
the tooth root. Thus, migration and eruption of teeth
can no longer occur, and the adaptability of the peri-
odontium is greatly impaired.
Sometimes localized ankylotic areas can be re-
moved and the integrity of the ligament restored
when periodontal ligament fibroblasts or their pro-
genitors are allowed to gain access to the root and
repopulate the area. These cells may come from the
adjacent periodontal ligament and perhaps also
from contiguous endosteal spaces in the alveolar
process but probably cannot differentiate from gin-
gival cell populations. Invasion of periodontal liga-
ment fibroblasts in an ankylosed site must be pre-
ceded by cells that have the capacity to resorb bone
and/or cementum (such as osteoclasts). A new peri-
odontal ligament space may be created at the cost
of the cementum (and sometimes part of the dentin)
and also of the alveolar process. Shortly thereafter,
this space is colonized by periodontal ligament cells
that form new periodontal ligament fibers, new ce-
mentum and a new alveolar wall in which the fibers
insert (201). Neither the nature of the signals that
initiate this repair process nor the molecular mech-
anism associated with cellular interactions is clearly
understood. However, it is known that under certain
conditions bone can be replaced by a functionally
oriented periodontal ligament of normal structure
and architecture, provided that the progenitors for
periodontal ligament fibroblasts can repopulate the
site. Moreover, masticatory function can accelerate
the resolution of ankylotic areas and restoration of
normal periodontal ligament width (3, 56, 199, 201).
The notion that periodontal regeneration specifi-
 The periodontal ligament: a unique, multifunctional connective tissue

Fig. 1. Light micrograph of human periodontal ligament Note the presence of Sharpey’s fibers in the bone (arrows).
showing the collagenous fiber meshwork interposed be- Azan stain, x250.
tween the root cementum (C) and the socket wall (B).

cally requires periodontal ligament cells is under- were subsequently inserted in artificial sockets pre-
lined by an experiment of Boyko et al. (401, who pared in edentulous areas of dog mandibles. Apart
seeded fibroblasts from either periodontal ligament from ankylotic areas, they observed after some time
or gingiva onto the surface of tooth roots, which areas with newly formed periodontal ligament fibers

21
Beertsen et al.

Fig. 2. Electron micrograph of periodontal ligament of ro-

dent molar showing fibroblasts oriented parallel to the
collagen fibers. Cementum (C); socket wall (B); nucleus
(N), x2000.
Fig. 3. High-power electron micrograph of collagen fibrils
in human periodontal ligament inserting into acellular
extrinsic fiber cementum [ x 25,000).

22
 The periodontal ligament: a unique, multifunctional connective tissue
Fig. 4. Light micrograph of distal aspect of human pre-
molar. Note the presence of many cement lines (arrow-
heads) in the socket wall (B) indicating sequential periods
inserted in newly formed cementum. This outcome
was observed only when periodontal ligament
fibroblasts were used as seeding cells: gingival cells
could not induce regeneration of the periodontal
ligament (98).
So far, no one has succeeded in establishing a new
and normally functioning periodontal ligament
without periodontal ligament cells, indicating that
these cells have specialized properties. Below we de-
scribe some of these properties in terms of structure,
architecture, functioning and regenerative potential,
with special emphasis on cellular and molecular
regulation. Given the scope of this chapter, we do
not cover every aspect of the biology and physiology
of the periodontal ligament. More complete reviews
of the periodontal ligament are available elsewhere
(31-33, 157).
Compositional and
structural aspects
The healthy periodontal ligament contains several
cell populations comprising fibroblasts, endothelial
cells, epithelial cell rests of Malassez, cells associated
with the sensory system, bone-associated cells and
of bone formation. Cementum (C); periodontal ligament
(P);Sharpey’s fibers (arrows). Hematoxylin and eosin
stain, x 130.
cementoblasts. The sensory system and the vascula-
ture of the periodontal ligament have been reviewed
(97, 101, 167). The predominant cell type is the
fibroblast (Fig. 21, which (in rodent molars) occupies
about 35% of the volume of the periodontal ligament
space (blood vessels excluded) (14) and in sheep in-
cisors about 20% (33). For adult human premolar
teeth a comparable cell density (ca 25%) has been
found (W. Beertsen, unpublished observations).
Matrix
The major fibers of the ligament are mainly colla-
genous in nature and consist of bundles of cross-
banded fibrils. In the adult human the individual fi-
brils are slightly thicker than those in several other
mammalian species, such as rodents (169), and
measure about 54-59 nm in diameter (107). They are
firmly anchored in the cementum and the bone by
Sharpey’s fibers (Fig. 3). Along appositional surfaces
of the socket wall (tension side), Sharpey’s fibers can
often be followed over great distances, far exceeding
the width of the ligament. As they traverse the bone
they cross many cement lines, representing sequen-
tial periods of active bone formation (Fig. 4). The
demarcating lines are relatively rich in the noncolla-
genous proteins osteopontin and bone sialoprotein
23

Fig. 5. High power electron micrographs of intercellular
junctions between periodontal ligament fibroblasts.
a. Gap junction. b. Adherence type junction. Collagen fi-
brils (c); oxytalan fiber (ox). a: ~ 1 0 0 , 0 0 0b:
; ~60,000.
(118, 119) which are thought to play a role in the
local regulation of bone metabolism. The great
length of the Sharpey’s fibers on the appositional
side of the socket wall suggests interstitial fiber
growth where the fiber bundles are incorporated into
the bone. Evidence for this arises from labeling
studies in mouse molars showing uptake of con-
siderable amounts of the collagen precursor ‘H-pro-
line (72).
The collagen fiber bundles in the ligament and the
fibers of Sharpey are mainly composed of interstitial
collagens I and I11 (43, 84, 196),which form banded
fibrils. Collagen V (one of the minor collagen mol-
ecules) is associated with these fibrils (1031, and is
either buried within the core of the fibrils (35) or is
found in the spaces between the fibril bundles (12).
All of these collagens are essential for the normal
architecture of the ligament, and none is restricted
to particular regions of the periodontal ligament (12,
48, 146). In addition to collagen V, the fibrous mesh-
work of the mature periodontal ligament has been
reported to contain several other minor collagens:
collagens VI and XI1 (12, 41, 53, 90, 170). Whereas
24
collagen VI is a microfibrillar component, not di-
rectly associated with the major banded collagen fi-
brils (but with the oxytalan fiber system (64)), colla-
gen XI1 belongs to the fibril-associated collagens
with interrupted triple helices that contribute to the
construction of the three-dimensional fibril arrange-
ment (130). Temporal and spatial expressions of col-
lagens I and XI1 in the remodeling periodontal liga-
ment during experimental tooth movement suggest
that collagen XI1 is closely associated with regenera-
tion of periodontal ligament function (89). Besides
collagens, several other proteins occur in the extra-
cellular matrix of the ligament that may have a re-
lationship with the macromolecular organization.
These include several proteoglycans (57, 81) and gly-
coproteins (such as undulin and fibronectin (207)).
Fibroblasts
The fibroblasts of the periodontal ligament are inter-
connected by numerous junctions which can be cat-
egorized as gap- and adherence-type junctions (Fig.
5) (18, 161). From observations on the rodent molar
ligament, it was estimated that each fibroblast is in
direct contact and communication with neighboring
cells by about 20 of such intercellular junctions (18).
Although communication between cells and mutual
interaction are facilitated via junctional complexes
and electrical coupling, chemical signals (paracrine
factors) are also believed to be important. The
fibroblasts in the ligament are oriented more or less
parallel to the collagen fibers (Fig. 21, whereas in
cross-sections they may exhibit a stellate appear-
ance, with cytoplasmic processes segregating indi-
vidual bundles of collagen fibers (Fig. 6). The cells
may attach to the collagen via a fibronexus-type of
attachment plaque (74, 85, 168) and are likely to
have the capacity to orient the extracellular matrix
(82). Together with their interaction with the colla-
genous framework, periodontal ligament fibroblasts
may also interact with an entirely distinct system,
the oxytalan fibers (21) which are predominantly
oriented in the apicocoronal direction, seemingly
unrelated to the collagen fiber system (Fig. 7). These
fibers were described initially by Fullmer (68) and
resemble pre-elastic fibers both histochemically (69,
145) and ultrastructurally. They contain a glyco-
protein with a molecular weight of 140 kDa (162) and
are associated with type VI collagen (64), one of the
minor collagens that can mediate cell attachment
(10). Although their resemblance to elastin suggests
that the oxytalan fibers may add to the elastic prop-
erties of the periodontal ligament, their function and
 The periodontal ligament: a unique, multifunctional connective tissue

extracellular matrix, properties that depend on the

level of expression of a-smooth muscle actin (6, 7).
Migration and contraction are in fact basic prop-
erties of many connective tissue cells and are of
great importance during developmental processes
and wound healing. For these activities the cyto-
skeletal apparatus is a prerequisite. Drugs that dis-
rupt the cytoskeleton also interfere with periodontal
ligament cell migration and eruption of rodent in-
cisors (27, 30, 45). Such drugs prevent the contrac-
tion of collagen gels in which periodontal ligament
fibroblasts are seeded and the movement of pieces
of dentin attached to it (6,27). Further, the migration
of periodontal ligament fibroblasts into wounded rat
molar ligament is impaired when the alkaloid colchi-
cine is locally administered to the rat lower jaw via

Fig. 6. Light micrograph of periodontal ligament of hu-

man premolar. Note that collagen fiber bundles (blue) are
cut transversely and segregated by cytoplasmic processes
of connective tissue cells (red).Azan stain, x 500.

cellular origin are unknown. Several functions have

been tentatively ascribed to this system, such as
regulation of vascular flow (165, 166) and facilitation
of fibroblast attachment and migration (21).

Fibroblast function
Although the migratory and contractile properties of
periodontal ligament fibroblasts have been docu-
mented in several animal and in uitro studies, little
if any is known about the migration and contraction
of cells in the healthy and functioning periodontal
ligament in humans. Migration of fibroblasts has
been shown in the periodontal ligament of continu-
ously erupting teeth (13, 14, 22, 206) and in teeth of
limited eruption during normal function (116, 136)
and during wound healing (76). There is also evi-
dence that cells in the vicinity of the alveolar wall
and the tooth surface may arise from precursor cells
that have divided in other locations (116). Peri-
odontal ligament fibroblasts, as most connective
tissue cells, are quite rich in cytoplasmic microfila-
ment systems which are indispensable for contrac-
Fig. 7. Oxytalan fibers (stained dark purple) running in
and movement (211 25J 134). et (259 the anico-occlusal direction. Note that their course does
26, 28) have shown that Periodontal ligament fibre- not ckespond with that of the collagen fibers in the peri-
blasts in vitro strongly contract and can orient their odontal ligament. Cementum (C), x 500.

25
Beertsen et al.
a mini-pump (W. Beertsen ?L T. Van den Bos, unpub-
lished). How the cells direct their migratory and con-
tractile activities and what signals are important are
largely unknown. Perhaps chemoattractants pro-
duced locally or by the hard tissue cells bordering
the ligament may have a role in these processes
(129). Directed migration of cells (and perhaps also
other functional activities) is associated with polarity
of organelles: the nucleus is usually in the trailing
portion of the cell, and the Golgi apparatus and cen-
triolar region are just in front of the nucleus towards
the leading edge of the cell, as is seen for instance
in migratory neutrophils (110). In this context, it is
intriguing that fibroblasts in rodent molar peri-
odontal ligament are polarized (19, 71). Although the
biological rationale of this phenomenon is still not
understood, it could have a bearing on directed ac-
tivities of the cells, such as directed migration or
contraction or directed synthesis and breakdown of
matrix components (19, 71, 73). Structural polarity is
perhaps also fundamental to fibril orientation in the
ligament, a phenomenon that to date has received
very little attention in periodontal literature. In fact,
the mechanisms by which fibrils and fibers in the
ligament (both collagen and oxytalan) are oriented
are unclear, except that fibroblasts are likely to be
important in this process.
Periodontal ligament fibroblasts have several other
characteristics which may help in distinguishing
them from other connective tissue cells in the body.
For instance, they appear to be rich in alkaline phos-
phatase activity (23,77,79).This enzyme is notable in
that it plays a key role in phosphate metabolism,
probably in mineralization processes (24, 184, 191)
and perhaps also in acellular (afibrillar) cementum
formation (78, 79). In this respect it is of interest that
alkaline phosphatase activity in rat molar periodontal
ligament shows a high correlation with acellular ce-
mentum thickness (79) and that in patients suffering
from hypophosphatasia - a recessive hereditary dis-
ease in humans characterized by very low concen-
trations of alkaline phosphatase in the blood and in
calcifyingtissues - cementum formation is greatly im-
paired (202).The enzyme is not restricted to cemento-
blasts and osteoblasts but is found in all periodontal
ligament fibroblasts, especially along their outer
plasma membrane face. Some alkaline phosphatase
(about 10%)is strongly associated with the extracellu-
lar collagenous fiber framework (80).What is not clear
from these data is why the entire periodontal ligament
does not mineralize in normal function in view of the
relatively high constitutive expression of alkaline
phosphatase activity.
Other cells
Apart from fibroblasts, the periodontal ligament
contains the epithelial rests of Malassez (Fig. 8)
which are derived from Hertwig’s epithelial root
sheath. The root sheath breaks up after initial root
formation and persists in the ligament as a wide-
meshed network (basket) of cells close to the tooth
surface. The cells have characteristics typical of epi-
thelial cells in that they are interconnected by de-
smosomes, contain tonofilaments and are sur-
rounded by a basal lamina. Although there has been
considerable speculation about their physiological
role, no evidence has emerged to support a specific
function (177, 200). In humans, rests of Malassez are
more frequent on the mesial side of molars than on
the distal side (188). In the mouse, rests of Malassez
are 3-4 times more frequent in the periodontal liga-
ment of the mesial root of the first molar as com-
pared with all other sites (200).In humans and other
mammals, their number tends to decrease with age
along all aspects of the root (147, 164, 188,200). Dur-
ing repair of molar periodontium in the mouse after
prolonged administration of the bisphosphonate 1-
hydroxyethylidene-1,l-bisphosphonate (which in-
duces localized ankylosis and narrowing of the liga-
ment space), re-formation of the periodontal liga-
ment does occur without the re-appearance of rests
of Malassez (200),suggesting that these cells do not
play a critical role in maintaining the normal width
of the ligament and in cementum formation and do
not prevent ankylosis and root resorption, as has
been suggested by several groups of workers (102,
105).
The cementoblast is of central importance in peri-
odontal ligament physiology and function. However,
since cementum is extensively dealt with elsewhere
in this volume (391, we will be very brief on this. For
a comprehensive review, see Schroeder (157) and
Freeman (67).
Although it is widely held that cementoblasts are
responsible for the formation of all types of ce-
mentum, there is only convincing evidence for their
involvement in the formation of cellular cementum,
which is usually found in the apical two thirds of the
root (37, 98, 157). The cementoblast forms a ce-
mentoid layer, which soon after its deposition un-
dergoes mineralization. Some of these cells are bur-
ied deeply in their own matrix and remain behind as
cementocytes, not unlike osteocytes in bone. It
seems doubtful whether specialized cementoblasts
occur adjacent to the acellular cementum at all de-
velopmental stages in all mammalian species. At
 The periodontal ligament: a unique, multifunctional connective tissue

Fig. 8. Rest of Malassez in rat molar periodontal ligament. Basal lamina (arrowheads);nucleus (N); X5000.

least in adult rodents (except for the growing ends
of the continuously erupting incisors) and in
humans no connective tissue cells, distinct from
periodontal ligament fibroblasts, can be seen in the
vicinity of acellular cementum layers. Only during
the initial stages of acellular cementum formation
in rat molars is there demonstrable evidence for a
specialized cell type (46). Cho & Garant (46) believe
that cementoblasts, derived from the ectomesenchy-
mal cells of the dental follicle, detach from the ce-
mentum surface and contribute to early periodontal
ligament fibroblast populations.
Matrix remodeling and
adaptability
Numerous studies have indicated that matrix pro-
teins of the periodontal ligament have an extremely
high turnover and remodeling rate, much higher
than in gingiva, skin and bone (171).Although these
studies have been performed in animal species other
than humans, there is evidence, albeit indirect, that
human periodontal ligament also has a high turn-
over rate and can easily adapt to changing local con-
ditions. This section discusses some of the cellular

27
Beertsen et at.
0 appear to occur throughout the periodontal liga-
---------
/'
-,
Fig. 9. Diagrammatic representation of collagen phago-
cytosis by fibroblasts. 1. Cytoplasmic protrusions of
fibroblast (Fi) surrounding a collagen fibril (co). 2. Lyso-
some (Ly) fusing with collagen-containing phagosome
and releasing its proteolytic content. 3. Phagolysosome
(PL). Reproduced with permission from Davidovitch Z, ed.
Biological mechanisms of tooth eruption and root resorp-
tion. Birmingham AL: EBSCO Media, 1988.
characteristics that are fundamental to the adapta-
bility of the periodontal ligament.
Both turnover and remodeling are characterized
by the coordinated breakdown and synthesis of
extracellular matrix components. In contrast to re-
modeling, turnover describes a process in which the
structural organization of the tissue remains un-
changed. During remodeling the three-dimensional
organization of the fiber meshwork is adapted to ac-
commodate for positional changes of the tooth in its
socket or changes in functional state (such as hypo-
function (15)). Both processes can occur simul-
taneously and may therefore be indistinguishable. It
has been suggested that the rapid remodeling is a
unique characteristic of the periodontal ligament
that relates to the adaptability of the periodontal
tissues (171).
Turnover and remodeling in the periodontal liga-
ment involve rapid synthesis and breakdown of mat-
rix components, most notably the collagenous
meshwork that stretches out between cementum
and bone. As mentioned above, the fibroblasts of the
periodontal ligament form a highly structured and
interconnected network, which strongly suggests the
coordinated action of cells. The collagen fibers of the
ligament form a meshwork of smaller fibers, each of
which is composed of unbranched collagen fibrils,
which may run from one fiber strand into another.
Although Sicher (163) suggested that remodeling in
the ligament is particularly confined to an inter-
mediate plexus in the mid-region of the periodontal
ligament, where fibers from the bone and fibers from
the tooth intermingle, turnover and remodeling in
teeth of limited eruption, like the molars of rodents,
ment from cementum to bone (16, 17, 150). Even
Sharpey's fibers during their incorporation into al-
veolar bone and cementum seem to be exposed to
considerable remodeling activity, as indicated by 3H-
proline incorporation (72).
In order to adapt to positional changes of teeth,
the fiber systems in the periodontal ligament must
be broken down. Because the collagen in the peri-
odontal ligament appears to form a complex mesh-
work, not unlike a stretched fishing net, breakdown
processes can occur at different sites without com-
promising tissue integrity. Thus, there is flexibility in
the system to permit adaptive changes by breaking
down short stretches of collagen fiber bundles or
single fibrils while leaving others intact. Although it
is still widely thought that degradation of collagen
requires the activity of collagenase in the extracellu-
lar space, biochemical studies have shed some doubt
on the role of this enzyme under steady state con-
ditions (60). First, despite the extremely short half
life of the collagens in the periodontal ligament
(1721, which has been estimated to be on the order
of several days in rodents (1711, collagenase has not
been detected in periodontal ligament tissues. Sec-
ond, substantial evidence indicates that collagen
degradation in the ligament occurs intracellularly
following a process of phagocytosis by periodontal
ligament fibroblasts (Fig. 9, 10) (21, 59-64, 66, 70,
104, 160, 180). Third, in vitro studies have shown
that collagen fibrils taken up by fibroblasts can be
broken down in lysosomal structures by the activity
of cysteine proteinases (61, 194) without the involve-
ment of collagenase (63). Lysosomes isolated from
pig periodontal ligament fibroblasts degrade colla-
gen and contain cathepsins B, D, H and N (195). In-
deed there is substantial evidence to indicate that
the intracellular and extracellular routes of collagen
breakdown constitute separate pathways (62, 192,
193). Studies by Svoboda et al. (179) have shown a
relationship between turnover rate of collagen in the
various tissues constituting the periodontium and
the amount of collagen ingested by fibroblasts, the
highest amount being found in the tissues with the
 The Deriodontul ligament: a unique, multifunctional connective tissue
Fig. 10. Collagen fibrils internalized by human peri-
odontal ligament fibroblasts (arrows).A. electron trans-
lucent vacuoles representing an early stage of collagen di-
highest turnover, most notably the periodontal liga-
ment.
What advantage could there be in degrading colla-
gen intracellularly instead of extracellularly? Phago-
cytosis allows a more precise and selective control
for the collagen fibers to be degraded, whereas the
release of extracellular collagenolytic enzymes af-
fords a more rapid, extensive degradation around
the cells, as observed during inflammation (173). Al-
though the work of Everts et al. (63) indicates that
collagenase is not important in collagen phago-
cytosis, the involvement of other members of the
gestion. B. The same, in cross section. C. Collagen-con-
taining vacuole fused with lysosome representing a later
stage in the digestive process. A, B, x 15,000; C, X60,OOO.
class of metalloproteinases (presumably the gela-
tinases) cannot be ruled out (62).
Although little is known about collagen degrada-
tion in the healthy human periodontium, electron
microscopic studies (71, 203) have shown that colla-
gen phagocytosis by fibroblasts is a normal feature
(Fig. 10). Thus, we adhere to the view that, in the
human periodontal ligament, collagen remodeling
and collagen breakdown do not differ essentially
from what is known about these processes in other
animal species.
Taken together, all these studies indicate that the
29
Beertsen et al.
periodontal ligament is characterized by a rapid
turnover and a high remodeling capacity. Under
steady-state conditions, collagen degradation in this
tissue is carried out via a process of collagen phago-
cytosis without the involvement of collagenase.
Force distribution and
dynamic role in bone remodeling
Periodontal ligament and alveolar bone cells are ex-
posed to physical forces in uivo in response to masti-
cation, speech, and orthodontic tooth movement.
Physiological loading of teeth or orthodontically in-
duced tooth movements involve remodeling of the
periodontal and gingival connective tissue matrices
(54, 148, 149, 182). As long as ankylosis does not oc-
cur, the general trend after application of physical
forces to teeth is preservation of the width of the
periodontal ligament, a remarkable process involv-
ing precisely controlled osteogenic resorption and
deposition at specific sites in the tissues. Although
the histological and some of the biochemical effects
of orthodontic force application have been de-
scribed (50, 148, 204), the mechanisms by which ap-
plied forces produce reactive changes in periodontal
ligament and bone cells are poorly understood.
Thus, while it is known that applied mechanical
force leads to more rapid bone remodeling in vivo
(151), knowledge of exactly how force distribution
from the periodontal ligament to the alveolar bone
regulates bone remodeling is limited. In spite of
these caveats, morphological observations of bone
and periodontal ligament after application of ap-
plied forces to mammalian teeth have lead to the
following general conclusions:
The periodontal ligament distributes applied
forces to the contiguous alveolar bone.
The direction, frequency, duration and size of the
forces determines in part the extent and rapidity
of bone remodeling.
When forces are applied to teeth devoid of a peri-
odontal ligament, the rate and extent of bone re-
modeling is very limited.
These conclusions suggest that the periodontal liga-
ment may be both the medium of force transfer and
the means by which alveolar bone - at least the wall
of the socket - remodels in response to applied
forces. Further, they strongly suggest that the peri-
odontal ligament is an irreplaceable tissue in the
context of force distribution and bone remodeling.
30
Indeed, osseointegrated implants, with no interven-
ing periodontal ligament, are used as immobile an-
chors because applied orthodontic forces induce
only very limited bone remodeling (83). However,
the periodontal ligament is a complex tissue con-
taining diverse mixtures of matrix proteins, cells and
vascular elements that are enclosed in very narrow,
yet precisely regulated dimensions. Thus, investigat-
ing directly and modeling the biophysical attributes
of the tissue are extremely difficult. In spite of these
limitations, progress over the last 5 years on force
transduction in biological systems has now been ap-
plied to bone remodeling and to the role of the peri-
odontal ligament. For example, Andersen et al. (2)
examined stress and strain levels and their distri-
bution within the periodontium in a model system
based on human autopsy material. This model sys-
tem permitted an estimate of the stress levels that
may be distributed across the periodontal ligament
under applied loads. Below we discuss data relating
to basic mechanisms of force transduction and how
the periodontal ligament may be involved in bone
remodeling. Notably, much of these data are from in
vitro studies or from in vivo rodent models, and the
direct application of these findings to the human
periodontal ligament is uncertain.
A wide range of approaches to model external
force application in vitro have been taken, including
subjecting cells to shear stress, hydrostatic pressure,
and strain of deformable substrates (126, 189). Cur-
rent evidence suggests that cells have a mechanism
to respond directly to mechanical forces by acti-
vation of mechanosensory signaling systems, includ-
ing adenylate cyclase and stretch-activated ion
channels, and by changes in cytoskeletal organiza-
tion. These alterations result in the generation of in-
tracellular second messengers such as intracellular
calcium ion concentration ([Ca2+Ii),inositol phos-
phate (IP3) and CAMP For example, [CaZ+lioscil-
lations are seen in periodontal cells responding to
substrate tension (6), and increased IP3 has been ob-
served after physical stretching of osteoblasts (44,
88). Intermediate-term responses to applied force
include generation of arachidonic acid metabolites.
For example, in uitro work shows that there is in-
creased prostaglandin release (176, 205), and in vivo
experiments of applied force to cat teeth also dem-
onstrated that there was increased immunoreactivity
for prostaglandin E2in expected tension areas of the
periodontal ligament (154). Interleukin- 1 p may also
be involved in periodontal ligament regulation of
bone remodeling in that cyclic-tension force causes
increased interleukin- 1 p production by human peri-
 The periodontal ligament: a unique, multifunctional connective tissue
odontal ligament cells (159), and a higher interleu-
kin- lj3 level was observed at tension sites of cat peri-
odontium in vivo (154). Longer-term responses to
mechanical loading in vitro include stimulation of
cell division (42, 491, altered collagen synthesis (88,
99), promotion of collagenase activity (96) and
stimulated release of transforming growth factor-p
(94). Intermittent pressure application to peri-
odontal ligament cells in vitro increases bone re-
sorption, which may involve the previously de-
scribed increased synthesis of prostaglandin Ez
(155). Collectively, these data indicate that there are
many potential routes by which applied loads to
periodontal ligament may lead either directly or in-
directly to alveolar bone remodeling. Current re-
search on mechanotransduction has focused on sig-
naling mechanisms and the identification of mech-
anosensors in periodontal ligament and bone cells.
Signaling mechanisms
The most rapid responses documented in peri-
odontal fibroblasts or osteoblasts subject to mech-
anical strain in vitro involve an elevation in [Ca2+Ii
(6,88),and changes in actin filament polymerization
(134), which implies a fundamental role for their
modulation of subsequent intracellular events. An
increase in calcium-channel or nonspecific cation-
channel conductance would permit such a rapid el-
evation in [Ca2+Iidue to influx down a strong elec-
trochemical gradient. Although earlier studies into
the mechanisms of physical force transduction in
periodontal tissues concentrated on the role of
piezoelectric charges, the vasculature, cytokines and
inflammatory mediators in regulating the response
of bone cells and fibroblasts to mechanical forces,
more recent studies have investigated the ability of
these cell types to respond directly to membrane
perturbation. Watson (197) has proposed that a
transducer of mechanical forces into intracellular
events would likely possess both multiple mem-
brane-spanning domains stabilized by electrostatic
attractions, and a functional association with the
cytoskeleton (such as actin microfilaments) to focus
the physical forces onto the mechanosensor. Other
authors suggest that the membrane itself has a low
bending modulus; physical forces such as shear
stress and direct stimulation may therefore cause a
conformational change of membrane-associated
protein complexes, leading to protein activation or
altered substrate-enzyme interactions (34).Stretch of
the cell membrane, which may be induced by cell
volume increase, can activate specific stretch acti-
vated ion channels, leading to an influx of calcium
ions down the electrochemical gradient (47, 51, 93).
Concurrent alterations in cell membrane structure
may expose and precipitate membrane phospho-
lipids (11, 197).The effect of physical forces on cyclic
AMP levels has also been well documented in ortho-
dontic tooth movement in vivo (44, 205) and follow-
ing cell stretching in vitro (124). However, the mech-
anism of the mechanosensitive transduction is still
speculative.
In addition to their structural role in cell shape
determination, microfilaments may transduce ap-
plied forces into intracellular signals (87), which is
perhaps mediated by local alterations in pressure or
volume. This, in turn, could alter molecular poly-
merization and subsequently modify cellular func-
tion (86, 87). Most models of cytoskeletal force trans-
duction focus on the effects of cytoskeletal interac-
tions with kinases, actin- and guanosine
triphosphate-binding proteins, and other regulatory
enzymes. With specific regard to intracellular cal-
cium metabolism, it has been proposed that micro-
filaments regulate stretch-activated ion channels
(153), functionally affect membrane enzymes and
substrates (such as phospholipase C and phosphoti-
dylinositol (52, 7511, and influence internal Ca2+ re-
lease by IPS (190). Microfilaments in turn are highly
regulated by [Ca2+Ii.
The ability of actin filaments to reorganize rapidly
in response to diverse external signals has been
demonstrated in cultured stromal cells in vitro. In
relation to physical stimuli, mechanical strain of at-
tached periodontal cells via a flexible substrate re-
duces F-actin content within 10 seconds, which is
followed by rapid polymerization (134). The poly-
merization state of the sub-membrane cortical actin
meshwork may affect the stretch-activated cation
channel current (153).
Collectively, these data indicate a dynamic, re-
ciprocal relationship between the activation of
membrane-localized cation-permeable channels
and the structure of the cytoskeleton and also that
stromal cells (and, in particular, the fibroblasts and
osteoblasts that populate the periodontal ligament)
have the necessary signaling and effector mechan-
isms to both sense applied physical force and to
mount a remodeling response. In the instance of the
periodontal ligament and the alveolar bone, these
cellular characteristics have an important conse-
quence: the periodontal ligament is an absolute re-
quirement for rapid remodeling of alveolar bone
when physical forces are applied to teeth.
31
Beertsen et al.
Regenerative potential
One of the major objectives of periodontal therapy
is to restore the lost fibrous attachment and bone
that occurs as a result of periodontitis and other
periodontal diseases. This objective requires the
existence of a coherent biological “program” to re-
store the destroyed connective tissue, form new ce-
mentum and bone and induce attachment of new
connective tissue fibers (9, 55, 143). This program
must also integrate these processes, and the peri-
odontal ligament likely plays a central, integrative
role in achieving periodontal regeneration. Con-
versely, it is difficult to conceive of how periodontal
ligament can be restored without the concomitant
production of new cementum and new alveolar
bone. Many reports indicate that restoration of de-
stroyed periodontal ligament is at least possible, al-
though their effectiveness is unclear and success is
unpredictable (108, 127, 128, 141, 142). Notably, re-
pair but not complete restoration of the lost peri-
odontal ligament is a frequent result.
Challenges of periodontal ligament regeneration
Peculiar to the healing of periodontitis lesions is the
fact that the various types of cementum are not re-
newed during normal function (158). Cementum
and the root surfaces of teeth that are exposed to
periodontal pathogens and their metabolites under-
go significant pathological alterations (1).Even after
debridement, the roots appear to favor the attach-
ment of epithelial cells compared with connective
tissue fibroblasts (186, 187). Although it may be ad-
vantageous to retain a long junctional epithelium for
protection against root resorption, it is not con-
ducive to connective tissue regeneration and ce-
mentum formation (185). As regeneration of peri-
odontal ligament necessitates direct contact be-
tween denuded root surfaces and regeneration-
competent connective tissue cells, it is of consider-
able importance that cells of the periodontal liga-
ment can interact with a biologically acceptable root
surface and not be blocked by epithelium. Indeed,
recent work (141) suggests the use of combinations
of extracellular matrix proteins to prevent specifi-
cally the migration of epithelium during periodontal
wound healing.
Cells contributing to regeneration
The periodontal ligament has long been suggested
to be of central importance in determining the
32
outcome of periodontal regenerative procedures
(120). In periodontal wound healing, the peri-
odontal ligament provides the following important
functions: 1) generates new fibers and continuity
of fibers from root to bone; 2) prevents apical mi-
gration of epithelium; 3) contributes cells to re-
store lost bone and cementum; 4) acts as a bio-
logical sensor and activator to regulate its own
width. The importance of the periodontal ligament
is perhaps best demonstrated by the considerable
interest that has been generated following the sug-
gestion of guided tissue regeneration (109). Briefly,
wound-healing experiments using occlusive mern-
branes to separate gingival from periodontal liga-
ment and bone compartments have indicated that
preferential colonization of periodontal wounds by
cells derived from the periodontal ligament may
result in enhanced healing (109). Although no de-
finitive studies on the origin of cells repopulating
wounded periodontal ligament have been under-
taken in humans, separate investigations using cell
kinetic (117) and cell culture methods (121) indi-
cate that periodontal ligament fibroblast popula-
tions in adult rodents are derived from precursor
cells of both the periodontal ligament and end-
osteal spaces. Although it is not possible to ex-
trapolate directly from the mouse or rat to the hu-
man, these data do suggest that there is consider-
able mixing of cell populations in periodontal
tissues. Thus, the basis for periodontal regenera-
tion by selective cell repopulation 191) remains
theoretical: it is not known whether anatomical
boundaries constrain cell populations with differ-
ent origins and phenotypes or whether separation
of cells by physical means will actually result in
the repopulation of a wound by a predetermined
phenotype. This point leads to an important ques-
tion: what cellular phenotypes are required for re-
generation? For example, several features of peri-
odontal ligament cells indicate that they may be-
long to a separate population distinct from
connective tissue cells in the gingival domain. In
addition to the features mentioned already, there is
type XI1 collagen expression (901, initiation of min-
eralized nodule formation in vitro (5) and vari-
ations of proliferative responses and matrix syn-
thesis profiles (111, 131, 174). None of these
markers alone is absolutely specific for periodontal
ligament cells, but collectively they indicate some
important functional attributes of these cells that
are important for regeneration. For example, the
expression of alkaline phosphatase by periodontal
ligament cells may be required for cementogenesis
 The periodontal ligament: a unique, multifunctional connective tissue
(78, 79). As the identity of the various periodontal
ligament cell populations is not certain, and as it
is recognized that several types of cell populations
inhabit this tissue, we will restrict our discussion
to the fibroblastic series. In the context of peri-
odontal ligament regeneration, as the formation of
functionally oriented fiber systems is a widely cited
indicator of periodontal regeneration (8, 55, 143), it
may be appropriate to examine this attribute as a
marker for regeneration-competent fibroblasts.
Fiber formation
In regenerating periodontal ligament, the fibroblast
population must first produce oriented collagen
bundles and then maintain the orientation of these
fibers during the development of normal function.
In uitro studies indicate that periodontal ligament
fibroblasts have the capacity to form fibrillar arrays
that are morphologically similar to their in uivo
counterparts (139, 140). Indeed periodontal fibro-
blasts have a constitutive capacity to orient them-
selves between two attachment points (29). Directed
cell migration is likely to play an important role in
this process (139) which, in turn, is affected by the
biochemical nature of the substrate at the attach-
ment points (106, 138).During migration, fibroblasts
are able to either deposit oriented extracellular mat-
rix or to orient existent extracellular matrix (25, 178).
Directed cell migration and matrix secretion (73) and
the existence of tension (137) have been demon-
strated in periodontal tissues during development
and homeostasis, thus providing support for in vitro
models. However, despite the interesting infor-
mation from these in uitro models, it is not under-
stood how these data apply to the regenerating peri-
odontal ligament, how fibrillar arrays around teeth
are maintained at such large distances from teeth or
how physical forces acting on teeth in viuo regulate
the formation of the arrays.
Regulation of periodontal ligament cell
differentiation
The hierarchy of periodontal ligament cell popula-
tions is not well understood. Cell kinetic experi-
ments in mice and rats (114, 116) have shown that
periodontal ligament fibroblast populations are a re-
newal cell system in steady state: the number of new
cells generated by mitosis is equal to the number of
cells lost through apoptosis and migration (115, 156).
Consequently, periodontal ligament fibroblast popu-
lations are renewal cell systems. In other renewal
systems, the most primitive cell is classified as a
stem cell, characterized by extensive self-renewal, re-
sponsiveness to regulatory factors, generation of
multiple types of different specialized cells (58) and
restriction to a specific niche within the tissue (144).
By analogy, progenitor cell populations of mouse
molar periodontal ligament are located adjacent to
blood vessels and exhibit some of these same fea-
tures of stem cells (76, 113).Although it is not known
whether the daughter cells of these paravascular
progenitors actually migrate and contribute to peri-
odontal ligament cell populations in steady state
(1161, they do repopulate wounded periodontal liga-
ment (76).The periodontal ligament may also be the
source of other cells, including tooth-related sites for
cementoblasts (1 16) and bone-related portions for
the osteoblasts.
The precise relationship between putative stem
cells in regenerating periodontal ligament and nor-
mally functioning (steady-state) tissues is not clear
(144). Studies in normal and wounded mouse peri-
odontal ligament (761, normal rat gingiva (135) and
inflamed monkey gingiva (123) have identified a
common paravascular location for fibroblast pro-
genitors. The differentiation control systems of
periodontal ligament cells are poorly understood
but are likely to be affected by the proximity of the
tooth root (100). Several different factors may coor-
dinate the control of periodontal ligament fibro-
blastic cell differentiation. Physical forces (1341,
lectins (132), cell shape and extracellular matrix
(198) and a variety of fibrogenic cytokines (95)
modulate the form and function of fibroblasts. As
there are no unambiguous differentiation markers
for fibroblast populations, there is little definitive
evidence on which populations of fibroblasts are
regulated by specific factors, particularly in the
periodontal ligament. Similarly, the locations of the
control points in the differentiation pathway are
unknown. However, in spite of these obstacles,
there has been considerable interest in assessing
the regulation of periodontal ligament function by
cytokines that are known to influence fibroblast
metabolism and wound healing (36, 112, 125, 152).
Collectively, these reports show that wound healing
cytokines, such as platelet-derived growth factor,
are mitogenic and that there is considerable inter-
action between cytokines when applied simul-
taneously (152, 192). There are no convincing data
yet that a single cytokine or multiple cytokines will
induce selective growth and differentiation of peri-
odontal ligament fibroblasts and not other stromal
cells in the wounded periodontium.
33
Beertsen et al.
Towards a rational approach for the regeneration
of periodontal ligament in uiuo
Short-term application of platelet-derived and insu-
lin-like growth factors can enhance new attachment
procedures in dogs (108, 133). A possible short-
coming in the application of cytokines to improve
periodontal wound healing is the nonspecific activi-
ties of some cytokines on different cell lineages in
time and space (65) and the rapid loss of topically
applied factors over time (108). Currently, we do not
have a detailed knowledge of the temporal distri-
bution of these substances, their concentrations or
how they interact with each other. In contrast, recent
experimental data on cementum-bound mitogenic,
migration and attachment factors (8, 122, 175) indi-
cate that informational molecules produced during
cementum formation may be “stored” in the ce-
mental matrix and that their regulation of peri-
odontal ligament regeneration is interfered with by
endotoxin and other molecules present on diseased
roots. Conceivably, the appropriately timed release
of cementum-bound proliferation and differen-
tiation factors may promote periodontal ligament re-
formation, although they certainly are not a pre-
requisite under every condition, since regenerative
cementum formation can occur directly over ex-
posed dentin surfaces in both humans and other
mammals (38).
Conclusion
Over the past decade, insight into the physiology of
the periodontal ligament and the properties of its
cells has increased. This insight has come from the
synthesis of research results from sources as varied
as in uiuo rodent models, in uitro studies of cells and
tissues and protein biochemistry. Based on these
studies, we conclude that the periodontal ligament
is a unique connective tissue: it cannot readily be
replaced by cell populations other than those that
have their origin in the ligament itself.
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