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Periodontology 2000, Vol. 24, 2000, 5672 Copyright C Munksgaard 2000Printed in Denmark All rights reserved
PERIODONTOLOGY 2000ISSN 0906-6713
Role of physical forces inregulating the form and function of
the periodontal ligamentCHRISTOPHER A. G. MCCULLOCH, PREDRAG LEKIC & MARC D. MCKEE
The periodontal ligament is a physically small but
functionally important tissue in tooth support, pro-
prioception and regulation of alveolar bone volume.
There is a long-standing and widespread interest in
the periodontal ligament as a model connectivetissue because of its rapid matrix turnover and its
ability to adapt to alterations of mechanical loading.
These features are mediated in part by heterogen-
eous cell populations that enable the roots of teeth
to maintain dynamic yet strong attachments to bone
in spite of highly variable applied force levels. The
remarkably precise maintenance of periodontal liga-
ment width in spite of these force levels or the res-
toration of the ligament space after surgical ablation
indicates the existence of highly effective regulatory
systems for measuring tissue domains and forinitiating localized matrix resorption and synthesis.
Constitutive adaptation to applied forces is mediated
in part by specific structural and regulatory proteins
expressed by periodontal ligament cells. As an ex-
ample of these adaptations we review recent evi-
dence of how cytoskeletal proteins mediate protec-
tive responses to applied force and how these re-
sponses may enable the cells to survive in a
mechanically active environment.
Background
The rapid growth of interest in endosseous implants
as surrogates for natural teeth could indicate that the
demise of the periodontal ligament is imminent. As
many types of implants do not employ a gomphosis
to provide support and attachment to the jaw bone,
what is the rationale for continued study of the peri-
odontal ligament? We suggest that the many inter-
esting functional and biological features of this
tissue, its basic role in the development and main-
56
tenance of the periodontium and its core function
in the healing of periodontal wounds underlines its
fundamental importance. Further, the rapid re-
modeling of extracellular matrix proteins in the peri-
odontal ligament is the basis for its utility as a modelsystem to study connective tissue homeostasis and
remodeling. This chapter considers two discrete yet
interrelated aspects of periodontal ligament physi-
ology that underline its unique biological character-
istics and its central role in tooth support: 1) homeo-
static mechanisms for preservation of tissue do-
mains; and 2) adaptational features to high load
bearing. For definitive reviews on the general fea-
tures of the periodontal ligament, we refer you to
Schroeder (71), Berkovitz & Moxham (10) and Berko-
vitz (11). Asin vivo
data on the regulation of peri-odontal ligament function in humans are limited,
much of the work discussed in this chapter relates
to teeth of limited eruption in rodents or in non-
human primates and in vitro work on human peri-
odontal cells. While these models provide interesting
insights into the human periodontal ligament in
vivo, they are by no means perfect surrogates and,
consequently, it is not appropriate or desirable to ex-
trapolate directly from these data to the human situ-
ation.
Periodontal ligament structure andorganization
The periodontal ligament is a complex, vascular, and
highly cellular soft connective tissue that attaches
the tooth roots to the inner wall of the alveolar bone.
In general, all ligaments and tendons consist of par-
allel bundles of collagen fibers; some contain an ad-
ditional network of elastic fibers (such as ligamenta
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Periodontal ligament homeostasis
nuchae and flava) (86). The mechanical strength of
tendons and ligaments derives largely from the mol-
ecular structure of the type I collagen molecule and
its ordered arrangement into fibers (47). The fibers
of the periodontal ligament form a meshwork similar
to a stretched fishing net that extends between the
cementum and the bone. The fibers are anchored by
their insertion into bone or cementum as Sharpeysfibers. The mechanism by which collagen fibers are
tethered at their extremities is likewise important in
defining the overall mechanical properties of the
tissue system as a whole (16).
The periodontal ligament is unique among the
various ligament and tendon systems of the body in
that it is the only ligament to span two distinct hard
tissues namely, tooth cementum and bone (Fig. 1).
While tooth cementum has been likened to bone in
terms of function and morphology, it nevertheless
possesses unique anatomical and structural prop-
erties and is positioned at a soft tissuehard tissueinterface that is absolutely critical to the process of
mastication. Such an important anchoring tissue for
attachment of the periodontal ligament to the tooth
is mirrored on the wall of the alveolus in that bone
exhibits similar features in regard to structure and
composition; likewise, the periodontal ligament has
the ability to attach periodontal ligament fibers to
the alveolar process of the mandible and maxilla (15,
37, 43). Collectively, this arrangement forms a sus-
pensory complex involving two hard tissues and the
intervening soft connective tissue comprising the
periodontal ligament.
As might be expected for any biological system
routinely receiving repetitive biomechanical strains,
here associated with mastication, there are special
metabolic requirements and an architectural tissue
design that facilitate the function of the periodontal
ligament. Not surprisingly, such a unique and dy-
namic connective tissue system involving multiple
tissues requires exquisite regulation at the cellular
level. Maintenance and remodeling of periodontal
ligament collagen fibers (2123, 81), together with
the embedding and calcification of their extremitiesto form Sharpeys fibers (37, 43), requires the con-
certed action of numerous cell types (25) and
multiple, synchronized signaling mechanisms to co-
ordinate these activities. Central to these integrated
activities is the periodontal ligament fibroblast,
whose responsibilities include the formation and re-
modeling of the periodontal ligament fibers, and
presumably a signaling system to maintain peri-
odontal ligament width and thickness across the soft
tissue boundary defined by this ligament (53). Such
57
a central role in the physiology of the periodontium
dictates the need for precise cellular organization
and cellular signaling. In the periodontal ligament,
cellular signals are, in part, mediated by the forces
transmitted to the fibroblasts via collagen fibrils with
which they are in direct contact (25). Although not
particularly well characterized at the molecular level
specifically for periodontal ligament fibroblasts,some reports are available (33, 39, 78). Cell-matrix
interactions between fibroblasts and the extracellu-
lar matrix have been extensively studied and are re-
viewed elsewhere (69).
At the tissue level, periodontal ligament fibro-
blasts are rather regularly dispersed throughout the
ligament and are generally oriented with their long
axes parallel to the direction of the collagen fibrils
(Fig. 1). By virtue of their ability to both synthesize
and shape the proteins of the extracellular matrix,
periodontal ligament fibroblasts generate an organ-
izational tissue pattern in which collagen fibrils formbundles that insert into the bone and tooth ce-
mentum as Sharpeys fibers (16, 43, 56). This struc-
ture conforms to the three-dimensional architecture
of the periodontal ligament in a very precise manner
(20). At the level of the fibroblast cell body, the nu-
cleus occupies a large percentage of the volume of
the cell, but the surrounding cytoplasm contains the
full complement of organelles necessary to effect
protein secretion (7) (Fig. 2). During development
and the initial formation of the periodontal liga-
ment, the cytoplasm-to-nucleus ratio is high, and
fibroblasts appear very active in terms of having an
extensive network of rough endoplasmic reticulum,
a well-developed Golgi apparatus and abundant se-
cretory granules containing predominantly type I
collagen molecules destined for export. The cells
also develop long and thin cytoplasmic extensions
that form three-dimensional veils that compart-
mentalize the collagen fibrils into fibers. At all levels
of the cell, intimate connections are established be-
tween the plasma membrane and individual colla-
gen fibrils. Presumably, these sites represent contact
points for integrin-matrix linkages such that strainoccurring in the ligament is transmitted to the cell,
and appropriate cell signaling cascades are activated
(27). Typically, the collagen fibrils of the periodontal
ligament are quite uniform in size but show some
minor variability with age and at different anatomi-
cal locations within the periodontal ligament.
Several studies have indicated that the extracellu-
lar matrix collagens of the periodontal ligament have
an extremely high turnover and remodeling rate,
much higher than in gingiva, skin and bone (77).
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Fig. 1. Light micrographs illustrating hard and soft tissue osteoblasts (Ob) at the crest of an inter-radicular alveolar
relationships in the periodontium. A. Low-magnification bone septum. D: dentin. DF. Age-related changes in peri-
image showing the histology of the periodontal ligament odontal ligament structure and organization. Early in its
(PL) and surrounding tissues. The periodontal ligament formation and just prior to tooth eruption (D), peri-
intervenes between the alveolar bone (AB) and the tooth odontal ligament formation by fibroblasts (F) forms co-
root, the latter consisting of dentin (D) and cementum. incident with extensive bone formation by osteoblastsOther nearby tissues include the pulp (P) of the tooth, (Ob) in each surrounding alveolus of the alveolar process
Hertwigs epithelial root sheath (HRS), marrow elements of the mandible and maxilla. Osteoblasts are positioned
(M) situated in the alveolar bone and the mandibular at the bone surface, whereas fibroblasts are dispersed
nerve (N). B, C. Higher magnification images showing throughout the developing periodontal ligament (PL).
periodontal ligament (PL) relationships with alveolar Collagen fibers are relatively small in diameter at this
bone (AB) and tooth. The collagen fibers of the peri- point, and their structure and insertions are not readily
odontal ligament span these two hard tissues, inserting visible. Capillaries (asterisks) are abundant at all stages of
into the matrices of bone on one side, and the cementum periodontal ligament development. At the time of tooth
(CEM) of the tooth on the other. Fibroblasts are abun- eruption (E), the periodontal ligament develops an obvi-
dantly dispersed throughout the periodontal ligament, as ous tissue organization such that collagen fibers are
are numerous capillaries. C illustrates bone formation by clearly defined and can be observed to insert as Sharpeys
58
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Periodontal ligament homeostasis
Turnover and remodeling in the periodontal liga-
ment imply synthesis and breakdown of matrix com-
ponents, particularly the collagenous fiber mesh-
work that extends between cementum and bone. As
indicated above, the collagen fibers of the peri-
odontal ligament form are critical for tooth support
and attachment to bone. They form a meshwork of
smaller fibers, each of which is composed of un-branched collagen fibrils that may run from one
fiber strand into another. It seems improbable that
one single fibril extends the entire dimensions of
tooth to bone, although there is no definitive evi-
dence for or against this view. Early work (72) sug-
gested that remodeling of the ligament is confined
largely to the mid-region of the periodontal ligament
where fibers from the bone and fibers from the tooth
interdigitate in an intermediate plexus. More re-
cent evidence suggests that this idea may not
necessarily be correct. Turnover and remodeling ac-
tivity in teeth of limited eruption, like the molars ofrodents, are found throughout the width of the peri-
odontal ligament from cementum to bone (6, 8, 66).
To adapt to changes of tooth position, the fiber sys-
tems in the periodontal ligament must be degraded
and new fibers synthesized. Since the periodontal
ligament is not made up of single strands of straight
collagen fibers but consists instead of a complex
meshwork, remodeling does not necessarily occur at
all sites synchronously. There is apparently some
fibers (arrows) into the alveolar bone (AB). Osteoblasts con-
form to this arrangement, and are clustered between the
insertion sites. Fibroblasts at this stage are abundant and
show a large amount of perinuclear cytoplasm housing the
extensive synthetic and secretory organelles expected for
such an active, collagen-producing cell. In association with
the organization of the periodontal ligament, bone re-
modeling occurs, as evidenced by the presence of cement
lines (CL) in the bone. In adult, mature periodontal liga-
ment of an erupted tooth (F), fibroblasts are more stellate
in appearance, and the volume of the periodontal ligament
that they occupy is less than at earlier stages, with extra-
cellular matrix (collagen fibers) being the predominant
component of the periodontal ligament. Although somesites in the alveolus show obvious insertions of Sharpeys
fibers (arrow) into bone, other surfaces of the alveolar bone
appear not to have obvious insertions of collagen fibers
into the bone. This observation may be related to the
timing of the bone remodeling cycle and the ability of resi-
dent fibroblasts and osteoblasts to act synergistically to
create locally new insertion sites. Nevertheless, a sufficient
number of biomechanically sound Sharpeys fibers exist at
any one time to accommodate the forces of mastication. D:
dentin. All samples are from post-natal rat periodontium
embedded in LR White and stained with toluidine blue.
Bars equal 50 mm.
59
flexibility in the system to permit adaptational
changes by breaking down short stretches of colla-
gen fiber bundles or single fibrils while leaving
others intact. This highly localized remodeling pro-
cess is undoubtedly facilitated by the phagocytosis
of collagen. Unlike the bulk removal of collagen that
is effected by extracellular matrix metalloprotein-
ases, collagen phagocytosis enables periodontal liga-ment fibroblasts to very precisely remove collagen
fibrils at specific sites (23).
While their roles in the periodontal ligament are
not yet clear, a number of reports have identified ad-
ditional extracellular matrix components including
collagen types V and VI, chondroitin sulfate, proteo-
glycans, fibronectin, tenascin and undulin (38, 48,
49, 91). An arborizing network of oxytalan fibers has
also been demonstrated in the periodontal ligament
and is most prominent in its occlusal half (7). In re-
lation to other ligaments and tendons, the peri-
odontal ligament is a highly vascularized tissue (13),and almost 10% of periodontal ligament volume in
rodent molar comprises blood vessels (53). This rela-
tively high blood vessel content may provide hydro-
dynamic damping to applied forces as well as pro-
vide high perfusion rates to this tissue.
Of particular importance to the function of the
periodontal ligament are its attachment points to
bone and tooth cementum (Fig. 3). At both sites, the
actual insertion of periodontal ligament fibers into
bone and cementum occurs as Sharpeys fibers, an
anatomical arrangement that represents the most
obvious means by which a tooth is retained in the
alveolus and in its occlusal plane. Once embedded in
either the wall of the alveolus or the tooth, Sharpeys
fibers calcify to a significant degree (36) and are as-
sociated with an abundance of noncollagenous pro-
teins commonly found in bone but also recently
identified in tooth cementum (17, 56). Notable
among these proteins are osteopontin and bone sial-
oprotein. Ultrastructural immunolabeling studies
using colloidal gold have demonstrated that high
levels of osteopontin accumulate at the insertion site
within the interfibrillar volume of the tissue (56), andit is thought that osteopontin and other proteins
contribute to the regulation of mineralization and to
tissue cohesion at these sites of elevated biomechan-
ical strain. Indeed, recent data directly demonstrate
that osteopontin is rapidly induced in alveolar bone
shortly after application of orthodontic forces to
teeth (84). A high concentration of noncollagenous
proteins relative to collagen could conceivably en-
dow unique and advantageous physical properties to
this critical hard tissuesoft tissue interface. Particu-
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McCulloch et al.
Fig. 2. Transmission electron micrographs and immuno- extensions (arrows) that envelop and define collagen fiber
cytochemical preparations illustrating the ultrastructure (CF) bundles. Nu, nucleus. B. Cross-sectional profiles of
of periodontal ligament fibroblasts, collagen fibrils, and the collagen fibrils (Coll) of mature periodontal ligament
cell-matrix relationships, and the intracellular pathway show them to be relatively uniform in diameter. C. Peri-
for collagen secretion. A. A transverse section through the odontal ligament fibroblasts show an extensive network of
periodontal ligament showing the stellate nature of peri- rough endoplasmic reticulum (rER) and a well-developed
odontal ligament fibroblasts (Fb) and their cytoplasmic Golgi apparatus with abundant stacked Golgi saccules
60
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Periodontal ligament homeostasis
larly for bone, remodeling activity successively
severs Sharpeys fibers to variable degrees as older
alveolar bone is replaced by new bone (37, 43). Con-
comitant with osteoclastic resorption, new connec-
tions are made in an asynchronous fashion such that
periodontal ligament fibers are continuously being
embedded in the alveolar wall.
Importantly, another architectural arrangement bywhich periodontal ligament fibers interface with the
bony surface has been identified. Here, Sharpeys
fibers are not readily identifiable as distinct bundles
within bone, but periodontal ligament fibers can be
observed to abruptly terminate at the bone surface
within a dark-staining band of variable, but generally
thin dimensions (37, 43). This band is rich in noncol-
lagenous proteins but is relatively poor in collagen
(Fig. 3). Based on its ultrastructure and protein com-
position as determined by immunogold cytochem-
istry, this band appears to represent a modified form
of cement line and is present at sites where collagenfibers of the periodontal ligament are directly ap-
posed to the bone surface and partly inserted into
this material. In all cases, this band of matrix is min-
eralized and rich in osteopontin, and may represent
a means by which a rapid connection of periodontal
ligament with the bone surface is established, al-
though it probably possesses less mechanical
strength. Full incorporation of the extremities of
periodontal ligament fibers at a later time would
likely require the concerted action of osteoblasts lin-
ing the alveolar wall.
(GS) showing typical, collagen-containing spherical dis-
tensions (SD) at their periphery. Transfer vesicles (TV) are
also common in this region of the cell. The plasma mem-
brane (PM) is in direct apposition with the collagen fibrils
(Coll) of the extracellular matrix. D, E. After post-embed-
ding, colloidal-gold immunocytochemistry on thin sec-
tions of periodontal ligament using antibodies raised
against mouse N-terminus collagen a1(I) to visualize in-
tracellular pathways for the production of type I collagenby periodontal ligament fibroblasts (Fb), gold particle im-
munolabeling indicates the presence of this protein in the
Golgi (G) region and secretory granules (SG) of these cells.
Ultimately, these a chains form the basis of the collagen
molecule, which assembles as collagen fibrils (Coll) in the
extracellular matrix of the periodontal ligament. rER:
rough endoplasmic reticulum; TV: transfer vesicles. Nu,
nucleus. Epon (AC) and LR White (D, E) sections of rat
periodontium stained with uranyl acetate and lead citrate.
Bars equal 5 mm (A) and 0.5 mm (B-E). Collagen antibody
courtesy of Paul Bornstein, Department of Biochemistry,
University of Washington, Seattle, USA.
61
Periodontal ligament cellpopulations
The healthy periodontal ligament contains several
discrete cell populations including fibroblasts, endo-
thelial cells, epithelial cell rests of Malassez, sensory
cells (such as Rufini-type end organ receptors),
osteogenic and osteoclastic cells and cementoblasts.The predominant cell type is the fibroblast, which
occupies about 30% of the volume of the periodontal
ligament space in rodents (8). The fibroblasts of the
periodontal ligament originate in part from the ecto-
mesenchyme of the investing layer of the dental pa-
pilla and from the dental follicle (82) and are differ-
ent from cells in other connective tissues in a num-
ber of respects. For example, the rapid degradation
of collagen by fibroblast phagocytosis is the basis for
the very fast turnover of collagen in the periodontal
ligament (23).
Although periodontal ligament cells are frequentlyconsidered as a homogeneous population, there are
some data indicating that the periodontal ligament
contains a variety of fibroblast populations with dif-
ferent functional characteristics (52). Whether these
subsets are derived from a single type of progenitor
cell is unknown. For example, the fibroblasts on the
bone side of the periodontal ligament exhibit more
abundant alkaline phosphatase activity than those
on the tooth side (32). Developmental differences
may also exist: Freeman & Ten Cate (24) and Ten
Cate (80) demonstrated that periodontal ligament
fibroblasts near the cementum are derived from the
ectomesenchymal cells of the investing layer of the
dental papilla, while fibroblasts near the alveolar
bone are derived from perivascular mesenchyme.
Cell kinetic experiments in rodent molar teeth
have shown that periodontal ligament cells comprise
a renewal system in steady state (51, 54). The rate of
cell renewal in the periodontal ligament is notable
for its rapidity and for the precise degree of regula-
tion in spatially discrete compartments. Periodontal
ligament progenitor cell populations undergo exten-
sive turnover in the maintenance of the steady stateand, in spite of extensive tooth drift or wounding,
many of these cells remain precisely located within
a narrow zone in the vicinity of small blood vessels
(30) and in the endosteal spaces of the adjacent al-
veolar bone (55). These cells proliferate, migrate and
ultimately produce more differentiated cells that can
synthesize bone, cementum and the extracellular
matrix of the periodontal ligament as has also been
shown in the developing periodontal ligament (62).
The generation of highly specialized cell popula-
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Fig. 3. Immunocytochemical preparations selected to il- gen fibers (CF) insert as Sharpeys fibers (SF) into alveolar
lustrate the distribution of the noncollagenous protein os- bone, where they are tethered into a mineralized matrix
teopontin (OPN) at periodontal ligament collagen fiber in- rich in noncollagenous protein and containing abundant
sertion sites into alveolar bone and tooth cementum. osteopontin. Intense immunolabeling for osteopontin
A, B. The extremities of periodontal ligament (PL) colla- commences where the collagen fibrils insert (arrows) into
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Periodontal ligament homeostasis
tions that can remodel and heal damaged tissues in a
temporally and spatially appropriate manner is
thought to be essential for the repopulation and dif-
ferentiation responses in healing periodontium fol-
lowing extirpation of the periodontal ligament (58).
The signals that regulate these processes include cell-
matrix and direct cell-cell interactions which are
known to control cell proliferation, differentiationand cell function (34). For example, periodontal liga-
ment cells produce cell adhesion proteins like vi-
tronectin, tenascin and undulin as well as several
integrin subunits (78). Adhesive and cell-to-cell inter-
actions may be conducted through systemically
acting regulators that act on specific cell types that ex-
press the appropriate cognate receptor (83) or
through intercellular functions such as gap-junction-
mediated calcium fluxes (88). Indeed, the fibroblasts
of the periodontal ligament are connected by special-
ized junctional complexes which include as gap junc-
tions (7, 9, 76). While paracrine and autocrine regula-tion of periodontal ligament function is undoubtedly
important in mechanical stress-induced differen-
tiation of periodontal ligament fibroblast function
(50), recent evidence on mechanical stress-induced
DNA synthesis (41) suggests that autocrine function
may not beas important as other systems that may in-
clude electrical coupling. Thus the connectivity of
cells in the periodontal ligament may be of consider-
able importance in terms of the propagation of mech-
anically induced signals. Notably, mechanical stimu-
lation of the periodontal ligament stimulates the ex-
an otherwise mineralized bone matrix, which here has
been decalcified to highlight the underlying organic ma-
trix. At the insertion site, osteopontin appears to accumu-
late throughout the inter-fibrillar spaces and is likely in-
volved in regulating calcification at these sites and/or par-
ticipating in the maintenance of tissue cohesion. CL,
cement line. C. Where periodontal ligament (PL) collagen
fibers (CF) do not obviously insert for any distance into
the alveolar bone, they frequently abruptly terminate on
the bone surface in an osteopontin-rich, layer of organic
material (arrows) that resembles a cement line typicallyfound in bone. This arrangement may serve as an alterna-
tive attachment mechanism for the adhesion of collagen
fibers to bone in the absence of Sharpeys fibers. Fb,
fibroblast. D. On the tooth surface, Sharpeys fibers (SF)
exist where collagen fibers (CF) of the periodontal liga-
ment (PL) insert into cementum (CEM). Like for bone, os-
teopontin is a prominent constituent of these insertion
sites in cementum, with immunolabeling commencing at
the edge of the mineralized cementum (arrows) and con-
tinuing internally. Fb, fibroblast. LR White sections of rat
periodontium stained with uranyl acetate and lead citrate.
Bars equal 1 mm (A) and 0.5 mm (BD).
63
Fig. 4. Photomicrographs of rat periodontium including
the periodontal ligament showing the preservation of the
ligament width in young (A: 6 weeks) and old (B: 1 year)
rats. The sections were immunostained for bone sialop-
rotein (brown staining). AB: alveolar bone; PL: peri-
odontal ligament; C: cementum; P: pulp. Bar equals 100
mm.
pression of connexin 43, an important protein in the
formation of gap junctions in periodontal ligament
cells (79).
Regulation of periodontal ligamentwidth
The cells, vascular elements and extracellular matrix
proteins of the periodontal ligament function collec-
tively to enable mammalian teeth of limited erup-tion to adjust their position while remaining firmly
attached to the bony socket. Indeed some of the
most interesting features of the periodontal ligament
are its adaptability to rapidly changing applied force
levels and the capacity to maintain its width at con-
stant dimensions throughout the lifetime of the ani-
mal (53) (Fig. 4). This preservation of periodontal
ligament width throughout mammalian lifetime is
an important measure of periodontal ligament
homeostasis and gives insight into the function of
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biological mechanisms that tightly regulate the met-
abolism and spatial locations of the cell populations
involved in the formation of bone, cementum and
the periodontal ligament. Cytokines and growth fac-
tors are important locally-acting regulators of cell
function and periodontal ligament cells are capable
of synthesizing and secreting some of these factors
(14, 18, 26). The ability of periodontal ligament cellsto synthesize and secrete a wide range of regulatory
molecules is an essential component of tissue re-
modeling and periodontal ligament homeostasis.
Notably, some of the transforming growth factor-b
isoforms synthesized by periodontal ligament cells
can induce mitogenic effects but can also dose-de-
pendently down-regulate osteoblastic differentiation
of periodontal ligament cells (18). On the other
hand, prostaglandins, which are also produced by
periodontal ligament cells, can inhibit mineralized
bone nodule formation and prevent mineralization
by periodontal ligament cells in vitro (60, 61). Peri-odontal ligament cells are also capable of regulating
bone formation by producing paracrine factors that
inhibit bone resorption (26). Conceivably, these mol-
ecules may modulate the osteogenic activity of peri-
odontal ligament cell populations and contribute to
the preservation of periodontal ligament width.
These types of cellular signaling systems may, there-
fore, be capable of accurately measuring and
maintaining the width of the periodontal ligament
in spite of high-amplitude physical forces during
mastication and despite the presence of osteogenic
cells within the whole width of the periodontal liga-
ment. Further, recent evidence has shown that the
pro-inflammatory cytokine interleukin-1 (75) and
one of the isoenzymes responsible for prostaglandin
synthesis (cyclooxygenase 2) (74) are induced by ap-
plied mechanical force on periodontal ligament cells
in vitro. As prostaglandins and interleukin-1 can
strongly induce matrix degradation, there is evi-
dently an important relationship between mechan-
ical forces, cytokine production and regulation of the
periodontal ligament space. The appropriate regula-
tion of these signaling systems is clinically importantsince the failure of homeostatic mechanisms to
regulate periodontal ligament width may lead to
tooth ankylosis and/or root resorption.
Experimental disruption ofperiodontal ligament homeostasis
Various experimental perturbations including des-
iccation (2), heat (46) and bisphosphonates (64, 87)
64
have been used to study homeostasis of periodontal
ligament width. These interventions rely in part on
the depletion of periodontal ligament cell popula-
tions or an apparent alteration of the differentiation
repertoire of periodontal ligament cells (44), disrup-
tion of periodontal ligament homeostasis and the
transient or permanent ingrowth of bone. Experi-
ments in dogs (40, 59), monkeys (1) and rodents (87)have shown that when periodontal ligament cells are
physically removed from the cementum or are per-
turbed by drugs such as the bisphosphonate 1-hy-
droxyethylidene-1,1-bisphosphonate, bone grows
into the periodontal ligament space and ankylosis
may occur. Although ankyloses can persist for long
periods of time, the tendency of the tooth root to
be resorbed and replaced by bone usually leads to
complete resorption over the long-term. As the peri-
odontal ligament is replaced with bone, propriocep-
tion is lost because pressure receptors in the peri-
odontal ligament are deleted or do not function cor-rectly. Further, the physiological drifting and
eruption of teeth can no longer occur and conse-
quently the ability of the teeth and periodontium to
adapt to altered force levels or directions of force is
greatly reduced.
To understand how periodontal ligament cell
populations restore their cellular and tissue do-
mains, appropriate model systems are required that
can provide insight into the origin of cells, their
regulation and differentiation. For this reason we
have used the rat periodontal window wound model
(Fig. 5) developed by Melcher (57) and modified by
Gould et al. (30). This model facilitates studies of
periodontal ligament homeostasis since precise por-
tions of the alveolar bone and the periodontal liga-
ment can be reproducibly deleted. Selective deletion
of the periodontal ligament and alveolar bone
causes a transient (60-day) disruption of the cellular
domains required to preserve homeostasis, thereby
providing a system to study the regulation of osteo-
genic cells by the adjacent periodontal ligament
cells.
To assess repopulation and differentiation of peri-odontal ligament cells in healing periodontal tissues,
we used combined 3H-thymidine labeling (31) and
immunostaining with a-smooth muscle actin, osteo-
pontin, alkaline phosphatase and bone sialoprotein
as differentiation markers of soft and mineralizing
connective tissue cell populations (45, 65). These
studies have shown that in contrast to ablation of
the periodontal ligament, preservation of the peri-
odontal ligament in the window wound model pro-
motes healing of the alveolar bone. Regardless of the
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Periodontal ligament homeostasis
type of wounding method a subset of periodontal
ligament cells express osteopontin, a widely recog-
nized but not wholly specific differentiation marker
of early bone formation. Immunostaining of peri-
odontal ligament cells for bone sialoprotein, a
marker of differentiated osteoblasts and cemento-
blasts, shows no staining reaction in periodontal
ligament cells under physiological or wounding con-ditions. The absence of this late marker of osteoblast
differentiation in repopulating periodontal ligament
demonstrates that, while a significant portion of
periodontal ligament cells may have osteogenic
characteristics (45, 67), these cells are blocked from
differentiating into osteoblasts. Consequently peri-
odontal ligament width is restored during the initial
healing phase (710 days, Fig. 6A) because osteo-
genic cells are unable to enter the mineralization
phase of osteogenic differentiation.
We have used bone morphogenetic protein-7 im-
plants in rat periodontal window wounds to probethe effect of this potent osteoinductive agent on
periodontal ligament cell differentiation (65). In
spite of the known ability of bone morphogenetic
proteins to induce ectopic bone formation in other
tissues (muscle) (85), periodontal ligament width is
preserved in healing periodontal tissues after
wounding. Bone morphogenetic protein-7 implants
are selective in promoting the proliferation and dif-
ferentiation of osteogenic cells but do not apparently
affect fibrogenic periodontal ligament cell popula-
tions, since the periodontal ligament width is un-
changed after administration of bone morphogen-
etic protein-7. However, administration of a bisphos-
phonate (1-hydroxyethylidene-1,1-bisphosphonate)
(44) reduces periodontal ligament width; this loss of
homeostasis could be the result of altered differen-
tiation of precursor cells in the periodontal ligament
and the recruitment of these cells into the osteo-
genic lineage. Notably, administration of 1-hydroxy-
ethylidene-1,1-bisphosphonate inhibits periodontal
ligament cell proliferative activity, reduces cell
counts and induces bone sialoprotein expression in
the body of the periodontal ligament, implying a dis-ruption of subsequent periodontal ligament cell dif-
ferentiation (Fig. 6B). Periodontal ligament width is
perturbed only after treatment with 1-hydroxyethyli-
dene-1,1-bisphosphonate for 2 weeks and occurs
after the reduction of periodontal ligament cell
counts, suggesting that a relatively small proportion
of non-osteogenic periodontal ligament cells is re-
quired for the maintenance of periodontal ligament
width.
To assess in more detail the requirement for speci-
65
Fig. 5. Diagrammatic representation of periodontal win-
dow wound through the buccal surface of the rat man-
dible. The wound model was originally developed by
Melcher (57) and modified later by Gould et al. (30). The
wound (W ) extirpates either alveolar bone alone or bone
and the periodontal ligament (PL). The regeneration of
the periodontal tissues following wounding provides an
excellent model to study the origin of the cells recoloniz-
ing extirpated periodontium and has been used exten-
sively for phenotyping of periodontal cells involved in re-
generation (44, 45) without microbial contamination from
the oral environment and without involvement of gingival
cells. AB: alveolar bone; C: cementum; P: pulp; D: dentine.
fic cell populations in tissue remodeling and the
preservation of periodontal ligament homeostasis,
we transplanted Lac-Z-positive murine periodontal
ligament cells into periodontal wounds of a Lac-Z-
negative animal (submitted manuscript). By trans-
planting previously characterized periodontal liga-
ment cells that express b-galactosidase (Lac-Z-posi-
tive cells) into periodontal wounds of rats not ex-
pressing this marker, we have examined the
differentiation in vivo of cells with a known initial
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McCulloch et al.
Fig. 6. A. Longitudinal section through rat periodontium ministered 1-hydroxyethylidene-1,1-bisphosphonate be-
including periodontal ligament (PL) and alveolar bone fore wounding. 1-hydroxyethylidene-1,1-bisphosphonate
(AB) stained for osteopontin. The periodontal ligament reduces cell counts in the body of the periodontal liga-
and bone were extirpated 10 days before sectioning. Note ment and causes the periodontal ligament width to
the staining for osteopontin in the newly formed bone shrink. Notably, periodontal ligament cells express bone
(NFB) and the restoration of normal periodontal ligament sialoprotein (blue staining in cells). The normal patterns
width. C: cementum. Bar equals 50 mm. B. In situ hybridi- of periodontal ligament homeostasis following wounding
zation for bone sialoprotein in a longitudinal section are lost, presumably because of switch of the periodontal
through rat periodontium as in A but animals were ad- ligament cell differentiation repertoire to an osteogenic
66
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Periodontal ligament homeostasis
phenotype. Interestingly, at the first post-wounding
time (7 days), transplanted cells are present through-
out the entire periodontal ligament and at bone re-
modeling sites (Fig. 6C). Transplanted cells located
in the periodontal ligament exhibit the same pheno-
typic expression as had been determined in in vitro
cell cultures (osteopontin-positive, bone sialoprot-
einnegative). However, at day 14 and 28 afterwounding, transplanted periodontal ligament cells
are not found in the periodontal ligament but in-
stead are located at bone remodeling sites (day 14)
or in the outer layer of the regenerating bone (day
28, Fig. 6D, E). At these later time periods, the Lac-
Z-positive cells express bone sialoprotein, a marker
of a differentiated osteogenic cell. These data show
that transplanted Lac-Z-positive/bone sialoprotein
negative cells can, when embedded in the peri-
odontal ligament, differentiate into osteogenic cells
and migrate into appropriate bony sites. Evidently,
there are well-regulated systems that ensure cellswith the capacity to differentiate into osteoblasts are
restricted to existing bony sites. This regulation may
depend in part on physical loading of the peri-
odontal ligament, since unloaded teeth exhibit a
narrow periodontal ligament space.
Force distribution andmediators of periodontal ligament
remodelingPeriodontal ligament and alveolar bone cells are ex-
posed to physical forces in vivoin response to masti-
cation, parafunction, speech and orthodontic tooth
movement. Physiological loading of teeth or ortho-
dontically induced tooth movements involve re-
modeling of the periodontal and gingival connective
tissue matrices. Although the histological and some
of the biochemical effects of orthodontic force appli-
cation have been described, the mechanisms by
which applied forces produce reactive changes in
periodontal ligament and bone cells are poorly
phenotype. Bar equals 20 mm. CE. Transplanted Lac-Z-
positive periodontal ligament cells transplanted into
wounded rat periodontium. At 7 days after wounding (C),
transplanted cells marked with Lac-Z by histochemistry
are present throughout the periodontal ligament. At 14
days after wounding (D) or 28 days after wounding (E),
cells are associated with the margin of the alveolar bone
(AB) and begin to express bone sialoprotein. Bar equals
20 mm.
67
understood. Thus, while it is known that applied
mechanical force leads to more rapid bone remodel-
ing in vivo (68), knowledge of exactly how force dis-
tribution from the periodontal ligament to the al-
veolar bone regulates bone remodeling is meager. In
spite of these limitations, morphological obser-
vations of bone and periodontal ligament after ap-
plication of applied forces to mammalian teeth havelead to the following general suppositions: 1) the
periodontal ligament distributes applied forces to
the contiguous alveolar bone; 2) the direction, fre-
quency, duration and size of the forces determines
in part the extent and rapidity of bone remodeling;
3) 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 ligament may be both the me-
dium of force transfer and the means by which al-
veolar bone remodels in response to applied forces.
Progress over the last 10 years on force transduc-tion in biological systems has now been applied to
bone remodeling and to the role of the periodontal
ligament in force adaptation. Komatsu et al. (42)
have used a simple animal model system to examine
stress-strain functions in which root sections from a
variety of animal species are extruded from the al-
veolar bone. They found that the organization of
periodontal ligament collagen at particular sites in
the periodontal ligament is closely related to the
load characteristics in vitro. Andersen et al. (3)
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. Mechanical forces can induce
fibronectin and collagen synthesis by periodontal
ligament cells in a strain magnitudedependent
fashion (35). These studies show in a reasonably di-
rect way that the metabolism and the organization
of the soft connective tissues of the periodontal liga-
ment are indeed modified by applied physical forces.
While applied loads may induce reactive changesin cells of the periodontium because of secondary
vascular and inflammatory effects, current evidence
suggests that periodontal ligament cells have a
mechanism to respond directly to mechanical forces
by activation of a wide variety of mechanosensory
signaling systems including adenylate cyclase,
stretch activated ion channels and by changes in
cytoskeletal organization. These alterations result in
the generation of intracellular second messengers
such as [Ca2]i, inositol 1,4,5-triphosphate and cyclic
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McCulloch et al.
adenosine monophosphate. For example, [Ca2]i os-
cillations are generated in periodontal cells re-
sponding to substrate tension (4), and increased in-
ositol 1,4,5-triphosphate has been observed after
stretching of osteoblasts (19). Intermediate-term re-
sponses to applied force may include generation of
arachadonic acid metabolites. Indeed, the recent
demonstration of increased COX-2 expression bystretching of periodontal ligament cells in vitro (74)
suggests a mechanism by which increased prosta-
glandin levels may be generated. Interleukin-1 may
also be involved in periodontal ligament regulation
of bone remodeling in that cyclic-tension force
causes increased interleukin-1 production by human
periodontal ligament cells (75). Aging may exert a
modulating effect on interleukin-1 production since
in vitro aged periodontal cells produce more in-
terleukin-1 when stretched than younger cells (73).
Longer-term responses to mechanical loading in
vitro may include stimulation of cell division, al-though this response in periodontal ligament fibro-
blasts is apparently not due to an autocrine regula-
tory mechanism (41). Increased collagen synthesis
(35) and stimulation of alkaline phosphatase activity
(89) are also force-induced downstream changes that
likely impact on altering the form and function of
loaded periodontal ligament. These data indicate
that there are many potential routes by which ap-
plied loads to the periodontal ligament may lead
either directly or indirectly to alveolar bone remodel-
ing. Currently, much of the ongoing work on mech-
anotransduction has focused on signaling mechan-
isms, and there is great interest in determining the
nature of the mechanosensors in periodontal liga-
ment and bone cells.
Some of the most rapid responses in periodontal
fibroblasts subjected to mechanical strain in vitroin-
volve an elevation in intracellular calcium ions
([Ca2]i) (4), and changes in actin filament poly-
merization (63), which implies a fundamental role
for their modulation of subsequent intracellular
events. An increase in calcium-channel or nonspec-
ific cation-channel conductance would permit arapid elevation in [Ca2]i due to ion influx down a
strong electrochemical gradient. While earlier
studies investigating the mechanisms of physical
force transduction in periodontal tissues concen-
trated on the role of piezoelectric charges, the vas-
culature, cytokines and inflammatory mediators in
regulating the response of bone cells and fibroblasts
to mechanical forces, more recent studies have in-
vestigated the ability of these cell types to respond
directly to membrane perturbation. In periodontal
68
ligament fibroblasts, stretch of the cell membrane
induced by hypoosmolar cell volume increase can
activate stretch activated calcium permeable ion
channels, leading to an influx of calcium ions (12).
The influx of calcium ions can then strongly induce
other effectors including those proteins that regulate
the cytoskeleton.
The ability of actin filaments to rapidly reorganizein 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 filamentous actin content within 10 seconds,
which is followed by rapid polymerization (63). The
polymerization state of the sub-membrane cortical
actin meshwork may then affect the mobility and
function of cell surface receptors and could also me-
diate stretch-activated cation channel current (70).
Mechanoprotection
The actin-dependent sensory and response elements
of stromal cells that are involved in mechanical sig-
nal transduction are beginning to be clarified. To
study the role of actin in mechanotransduction we
have described a collagen-magnetic bead model in
which application of well-defined forces to integrins
induces an immediate (1 second) calcium influx
(28). We used this model to determine the role of
calcium ions and tyrosine-phosphorylation in the
regulation of force-mediated actin assembly and the
resulting change in membrane rigidity (27). Colla-
gen-beads were bound to periodontal cells through
the focal adhesion-associated proteins talin, vincul-
in, a2-integrin and b-actin, indicating that force ap-
plication was mediated through cytoskeletal ele-
ments. When force (2 N/m2) was applied to collagen
beads, confocal microscopy showed a marked verti-
cal extension of the cell, which was counteracted by
an actin-mediated retraction. Immunoblotting
showed that force application induced F-actin ac-
cumulation at the bead-membrane complex, butvinculin, talin and a2-integrin remained unchanged.
Atomic force microscopy showed that membrane
rigidity increased 6-fold in the vicinity of beads ex-
posed to force. Force also induced tyrosine phos-
phorylation of several cytoplasmic proteins, includ-
ing paxillin. The force-induced actin accumulation
was blocked in cells loaded with the intracellular cal-
cium chelator BAPTA/AM or in cells pre-incubated
with genistein, an inhibitor of tyrosine phosphoryla-
tion. Repeated force application progressively inhib-
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Periodontal ligament homeostasis
ited the amplitude of force-induced calcium ion flux.
As force-induced actin reorganization was depend-
ent on calcium and tyrosine phosphorylation, and
as progressive increases of filamentous actin in the
submembrane cortex were correlated with increased
membrane rigidity and dampened calcium influx,
we suggest that cortical actin regulates stretch-acti-
vated cation permeable channel activity and pro-vides a desensitization mechanism for cells exposed
to repeated long-term mechanical stimuli. Thus, the
actin response may be cytoprotective since it
counteracts the initial force-mediated membrane ex-
tension and potentially strengthens cytoskeletal in-
tegrity at force-transfer points.
It seems self-evident that to survive in a mechan-
ically active environment, cells must adapt to vari-
ations of applied membrane tension. Part of this ad-
aptation involves sensing increases in extracellular
tension, maintaining contact with extracellular ma-
trix ligands and preventing irreversible membranedisruptions. Again with the use of the collagen-
coated magnetic bead model to apply forces directly
to the actin cytoskeleton through integrin receptors,
we investigated how the cytoskeleton reorganizes in
response to increased membrane tension. We found
that by a calcium-dependent mechanism, human
periodontal fibroblasts reinforce locally their con-
nection with extracellular adhesion sites (collagen-
coated beads) by recruiting actin binding protein-
280 into the cortical adhesion complexes (29). Actin
binding protein-280 was phosphorylated on serine
residues as a result of force application. This phos-
phorylation and the force-induced actin reorganiza-
tion were inhibited by bisindoylmaleimide, indi-
cating a role for protein kinase C isoforms. In a hu-
man myeloma cell line that does not express actin
binding protein-280, actin accumulation could not
be induced by force, while in stable transfectants ex-
pressing actin binding protein-280, force induced
actin accumulation similarly to human fibroblasts.
Cortical actin assembly evidently played an import-
ant role in regulating the activity of stretch-activated,
calcium permeable channels since sustained forceapplication desensitized these channels to sub-
sequent force applications and the decrease in
stretch sensitivity was reversed after treatment with
cytochalasin D. Further, in comparison to actin
binding protein-280-positive cells, actin binding
protein-280-deficient cells exhibited an almost two-
fold increase in stretch-activated channel activity
and significantly less (50% compared to 90% in actin
binding protein-280-positive cells) channel desensit-
ization following prolonged force application. Actin
69
binding protein-280-deficient cells showed a 90%
increase in cell death compared to actin binding
protein-280-positive cells (30% increase) after force
application, indicating a potential mechanoprotec-
tive role for force-induced actin binding protein-
280/actin reorganization. We suggest that actin bind-
ing protein-280 plays an important role in mechano-
protection by: 1) reinforcing the membrane cortexand thereby preventing force-induced membrane
disruption; 2) increasing the strength of cytoskeletal
links to the extracellular matrix; and 3) desensitizing
stretch activated ion channel activity.
Conclusions
Collectively, these data, which are largely from in vi-
tro investigations, indicate that stromal cells, and in
particular the fibroblasts and osteoblasts that popu-
late the periodontal ligament, have the necessarysignaling and effector mechanisms to both sense ap-
plied physical force and to mount a stream of re-
sponses which serve to maintain periodontal liga-
ment width and preserve cell viability. In the in-
stance of the periodontal ligament and the alveolar
bone these cellular characteristics have an important
consequence: the periodontal ligament is an abso-
lute requirement for rapid remodeling of alveolar
bone when physical forces are applied to teeth. This
requirement may be critically important for the
maintenance of alveolar bone volume following
tooth extraction since the loss of the periodontal
ligament terminates the mechanotransduction sig-
nals required for bone homeostasis.
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