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in Oral Biology & Medicine

Cementum and Periodontal Wound Healing and Regeneration

Wojciech J. Grzesik and A.S. Narayanan
CROBM 2002 13: 474
DOI: 10.1177/154411130201300605
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CEMENTUM AND PERIODONTAL WOUND

HEALING AND REGENERATION
Wojciech J. Grzesik1
A.S. Narayanan2*
1Dental

Research Center, CB#7455, University of North Carolina, Chapel Hill, NC 27599-7455; and 2Department of Pathology, Box 357470, University of Washington School of Medicine, Seattle,
WA 98195-7470; *corresponding author, sampath@u.washington.edu
ABSTRACT: The extracellular matrix (ECM) of cementum resembles other mineralized tissues in composition; however,

its physiology is unique, and it contains molecules that have not been detected in other tissues. Cementum components
influence the activities of periodontal cells, and they manifest selectivity toward some periodontal cell types over others.
In light of emerging evidence that the ECM determines how cells respond to environmental stimuli, we hypothesize that
the local environment of the cementum matrix plays a pivotal role in maintaining the homeostasis of cementum under
healthy conditions. The structural integrity and biochemical composition of the cementum matrix are severely compromised in periodontal disease, and the provisional matrix generated during periodontal healing is different from that of
cementum. We propose that, for new cementum and attachment formation during periodontal regeneration, the local
environment must be conducive for the recruitment and function of cementum-forming cells, and that the wound matrix
is favorable for repair rather than regeneration. How cementum components may regulate and participate in cementum
regeneration, possible new regenerative therapies using these principles, and models of cementoblastic cells are discussed.
Key words. Cementum, extracellular matrix, periodontium, periodontal regeneration, healing.

Introduction

eriodontium" refers to the tissues that collectively invest

and support the teeth, and consists of the gingiva, periodontal ligament, alveolar bone, and cementum. The structure
and composition of the periodontium are affected in many
acquired and heritable diseases, and the most significant
among these is periodontal disease. The hallmarks of periodontal disease are destruction of soft connective tissues, bone
loss, and loss of connective tissue attachment to cementum;
these alterations, if left untreated, lead to tooth loss. The aim of
periodontal therapy is to regenerate and restore the various
periodontal components affected by disease to their original
form, function, and consistency (Aukhil, 1991; Garrett, 1996).
Regeneration requires restoration of alveolar bone height to
the cemento-enamel junction, regeneration of gingival connective tissue destroyed by inflammation, formation of new acellular extrinsic fiber cementum on previously exposed root surfaces, synthesis of Sharpey's fibers and their insertion into root
surfaces, and re-establishing epithelial seal at the coronal portion. New therapeutic approaches available to achieve these
objectives include use of barrier membranes for guided tissue
regeneration, and applying growth factors and enamel matrix
proteins to root surfaces; however, the effectiveness of these
approaches is not predictable, especially on new cementum
and attachment formation. These approaches and the principles behind them have been reviewed recently in several
excellent publications (Cochran and Wozney, 1999; MacNeil
and Somerman, 1999; Wikesj and Selvig, 1999; Bartold et al.,
2000); therefore, they will not be discussed here. Cementum is
the site where soft-tissue attachment has to be re-established,

474

and cementum matrix is a rich source of many growth factors

which influence the activities of various periodontal cell types
(Narayanan and Bartold, 1996; Saygin et al., 2000); therefore, in
this review we will examine how the cementum can play a
regulatory role in periodontal regeneration. In light of emerging evidence showing that local environment is a major regulator of how cells respond to environmental cues and signals
(Sastry and Horwitz, 1996; McKay, 1997; Michalopoulos and
DeFrances, 1997), we believe that relatively little emphasis has
been placed on the role of cementum in periodontal regeneration. Our goal is to highlight the manner by which cementum
constituents can participate in and regulate periodontal regeneration, and to discuss how these principles can be extrapolated to regenerative therapy of the periodontium.

Wound-healing Events
To facilitate discussion on the possible role of cementum in
periodontal regeneration, we will first briefly review the events
and molecules involved in wound healing. Wound healing
involves three overlapping phases that are interdependent
(Clark, 1996). The general principles and events have been
characterized with the use of cutaneous wound models, and
these apply to healing of surgical and diseased periodontal
wounds as well. Traumatic injury damages blood vessels and
causes hemorrhage and extravasation of blood, and a blood
clot is formed. The blood coagulation process and activated
complement pathway generate many polypeptide mediators,
and the blood clot serves as a provisional matrix for the migration of inflammatory cells. Neutrophils and monocytes cleanse
the wound of bacteria, foreign particles, and dead tissue, and

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macrophages and platelets secrete several polypeptide mediators that direct and regulate the activities of various cells participating in healing.
Re-epithelialization begins within hours of injury, when
epithelial cells near the wound margin migrate to the wound
site. The number of epithelial cells increases at the wound due
to migration and proliferation, and eventually the breach in
epithelium is sealed. During this time, angiogenesis and synthesis of collagens and other ECM components become active,
and the clot is replaced by "granulation tissue". The wound is
filled with activated fibroblasts, and a proportion of these cells
is transformed into myofibroblasts. Appearance of the myofibroblasts corresponds to the beginning of connective tissue compaction, and the wound contracts. The wound contraction
shrinks the wound size and brings the wound margins toward
one another (Singer and Clark, 1999). During the ensuing "tissue remodeling phase", the granulation tissue matrix is
replaced with fresh connective tissue, and new blood vessels
disappear through degradation and apoptosis. A fibrous scar
replaces the wound when regeneration is not possible. During
the following weeks and months, remodeling continues, and
up to 70% of original tissue strength is restored (Clark, 1996;
Singer and Clark, 1999).
Wound healing involves migration, adhesion, proliferation, and differentiation of several cell types. All these activities are triggered when polypeptide mediators bind to their
cell-surface receptors and when integrins bind to ECM components. Different growth factors regulate different cell functions. For example, transforming growth factor (TGF)-a, a
member of the epidermal growth factor (EGF) family, and
heparin-binding EGF (HB-EGF), keratinocyte growth factor
(KGF, also called FGF-7), and TGF-b and their receptors are
involved in re-epithelialization. The expression of KGF is
enhanced 100-fold 24 hours after injury (Martin, 1997). FGF-1
and -2 (acidic and basic FGF), vascular endothelial cell growth
factor (VEGF), TGF-b, angiogenin, angiotropin, angiopoietins,
and hepatocyte growth factor are strongly angiogenic, whereas thrombospondins and angiostatin inhibit angiogenesis
(Conway et al., 2001). Platelet-derived growth factor (PDGF),
which is mitogenic to fibroblasts and other mesenchymal cells,
is an important growth factor in wounds, and gingival epithelium is a rich source of this growth factor in gingival wounds
(Green et al., 1997). Fibroblast collagen synthesis is activated
by TGF-b and connective tissue growth factor (CTGF). The
TGF-b also appears to induce the transformation of fibroblasts
into myofibroblasts, and TGF-b and PDGF participate in
wound contraction.
Cell migration and adhesion require active participation of
integrins. For instance, migration of epithelial cells requires
dissolution of a6b4 integrin, the receptor that mediates the
attachment of hemidesmosomes and laminin. The receptors to
fibronectin and tenascin, a5b1 and avb6 integrins, and vitronectin receptor avb5 are expressed, and relocalization of type
I collagen receptor a2b1 occurs. The appearance of integrins
that bind to fibrin and fibronectin seems to be rate-limiting for
granulation tissue formation, and at the top of sprouting capillaries, endothelial cells actively express the integrin a5b3.
Integrins are also required for wound contraction.
In addition to changes in integrin expression, proteolysis is
necessary for cell migration through the fibrin clot and matrix
and for tissue remodeling. Inflammatory cells and most other
cells produce several different matrix metalloproteinases
13(6):474-484 (2002)

(MMPs) during wound healing. In cutaneous wounds, keratinocyte migration at the leading edge requires the fibrinolytic enzyme plasmin, and tissue-type plasminogen activator
(tPA) and urokinase-type plasminogen activator (uPA) activate
the plasmin. The expression of uPA, tPA, MMP-1 (also called
interstitial collagenase), MMP-9 (gelatinase B), and MMP-10
(stromelysin-2) is up-regulated at the wound margin.
Keratinocyte movement requires MMP-1 expression, and this
enzyme also facilitates cell directionality; the MMP-1 expression is turned off once re-epithelialization is complete (Parks,
1999). The MMPs also participate in remodeling of the granulation tissue matrix. Although there is considerable redundancy in the substrate specificity of MMPs, differences in their
affinities to different substrates and their compartmentalization
determine the course of their action and the composition of
matrix laid down.
Several "master genes" participate in the wound-healing
process. These genes code for proteins that control body plan
formation during embryogenesis and development, and
include homologues of TGF-b and BMPs and transcription factors encoded by "homeobox genes". The latter are characterized
by the presence of a common 61-amino-acid DNA-binding
motif called "homeodomain". These proteins regulate the
expression of lineage-specific genes during normal embryonic
development, and control pattern formation and cell fate and
identity. In adult eukaryotic cells and during wound healing,
the master genes participate in cell growth and differentiation,
apoptosis, and cell-cell and cell-ECM interactions (Srebrow et
al., 1998; Cillo et al., 2001). Several homeobox genes are
involved in vascular remodeling and angiogenesis (Gorski and
Walsh, 2000). The expression of master genes is regulated
through interactions among cytokines, growth factors, ECM,
and adhesion molecules, and growth factors that regulate their
expression include TGF-b, BMPs, and FGFs. For example, the
muscle segment homeobox gene MSX and "paired" PAX are
regulated by FGFs and BMPs. Altered expression of these
genes is associated with neoplastic and metabolic diseases
(Cillo et al., 2001). Programmed expression of master genes is
likely to be essential for differentiation programs associated
with wound healing, and aberrations in their regulation may
lead to wound repair rather than regeneration.

Factors that Determine Whether Wounds Heal

by Regeneration or Repair
Although the extent of injury and the amount of lost tissue that
must be filled are important determinants, whether a damaged
tissue heals by regeneration or is repaired by fibrous scar
depends upon two crucial factors: (1) the availability of needed
cell type(s), and (2) the presence or absence of the cues and signals necessary to recruit and stimulate these cells. These factors
are not mutually exclusive. The signals are provided by diffusible factors such as growth factors and cytokines; however,
the ECM regulates how cells respond to the signals.
In most vertebrates, the capacity to regenerate is limited to
a few tissues, such as liver and bone. In these tissues, regeneration may mimic events of embryonic differentiation of multipotential stem cells. However, mammalian tissues constituted
by "permanent cells" such as neurons and cardiac myocytes
usually heal by repair. One reason for the failure of these tissues to regenerate was believed to be due to the absence of
appropriate stem or progenitor cells. However, many adult tissues undergo self-renewal; therefore, they must presumably

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Differentiation and survival can also involve cross-talk with

other cells of the same and different cell types (Denker et al.,
1999; Fuchs and Segre, 2000). Examples for the latter include
mesenchymal cues for epithelial differentiation, and vice versa.
Substances present in the circulation as well as those in the
local environment provide stimulus and directional signals for
cells to migrate. These substances, which include growth factors, other soluble mediators, and ECM components, also promote cell division and differentiation, and more than 2000 such
molecules have been purified or cloned so far (Fuchs and Segre,
2000). During the course of wound healing, the spectrum and
concentration of molecules change continuously. More than
one substance is present in the local microenvironment, and the
combined effect of these molecules may be additive, synergistic, or antagonistic.

Regulation by the Local Environment

Figure 1. Stages in the differentiation of a stem cell. Embryonic stem

cells are nearly totipotent, whereas a stem cell in an adult tissue may
be pluripotent (for example, hematopoietic stem cell) or unipotent
(epithelial cell, for example). An adult tissue may contain the stem
cells and precursor cells separated from full differentiation by one or
several steps. The periodontal ligament has been shown to contain
precursor cells or progenitors to cementoblasts (Pitaru et al., 1994).
The differentiated cell may also consist of subpopulations of the same
cell type (McCulloch and Bordin, 1991; Fries et al., 1994).

contain a life-long population of relatively slow-cycling (stem)

cells. Indeed, recent evidence has shown that multipotent stem
cells are present in non-regenerating adult tissues, including
the brain (McKay, 1997). The stem cells, which, in nonhematopoietic tissues, are referred to as mesenchymal stem
cells or marrow stromal cells, can differentiate into many cell
types. Embryonic stem cells are almost totipotent and malleable, and the diversification of the embryonic cells into cell
types of their destined fate is largely complete shortly after
development is complete. On the other hand, stem cells from
adult tissues are relatively less malleable, and they may be
pluripotent (hematopoietic and neuronal stem cells) or unipotent (epithelial stem cells are committed to differentiate into
epithelial cells) (Krebsbach et al., 1999; Fuchs and Segre, 2000).
When a stem cell undergoes commitment to differentiate, it
often proliferates rapidly first and then differentiates, and the
differentiation to the final fate may occur in several stages (Fig.
1). These cells presumably respond to specific environmental
signals to generate new stem cells or differentiated cells. Under
normal homeostasis, differentiation of the stem cells is most
likely triggered by instructive and stimulatory signals provided by the (local) environment, which consists of the ECM and
growth factors sequestered in the matrix (Fig. 2, see later).

476

The ECM is a multicomponent three-dimensional structure

composed of collagens, fibronectin, elastin, other non-collagenous proteins, and proteoglycans. It serves as substratum for
cell adhesion and promotes cell spreading and cytoskeletal
organization (Folkman and Moscona, 1978; Bhmer et al., 1996;
Assoian and Zhu, 1997). The ECM determines the three-dimensional cell architecture and transmits and translates external
mechanical and tensional forces to appropriate response signals (Huang and Ingber, 2000). Adhesion to ECM is essential
for cell cycle transit by anchorage-dependent cells. The ECM
also regulates gene expression of growth factors, growth factor
receptors, and other proteins, and determines the outcome of a
cell's response to growth factors. For example, in the presence
of ECM, cell division is suppressed, and cells differentiate
(Adams and Watt, 1993; Juliano and Haskill, 1993; Gumbiner,
1996; Sastry and Horwitz, 1996).
Cells bind to the ECM through integrins, and the binding
initiates a cascade of signaling reactions. Signaling reactions
activated include tyrosine phosphorylation of focal adhesion
kinase (FAK) and other signaling proteins, activation of mitogen-activated protein kinase (MAP kinase) cascade, expression
of c-fos, and elevation of certain cyclin levels (Juliano and
Haskill, 1993; Yamada and Miyamoto, 1995; Assoian, 1997;
Assoian and Zhu, 1997; Lewis et al., 1998). Signaling pathways
induced by ECM components cooperate with those activated
by growth factors in mediating their biological functions, and
both integrin- and growth-factor-induced signals are necessary
for expression of G1 cyclins and cell cycle progression from
G0/G1 to S-phase. During G1, the expression of D1 and A
cyclins and activation of cyclin-dependent kinase (cdk)-2
require adhesion-mediated integrin signals (Guadagno et al.,
1993; Fang et al., 1996; Zhu et al., 1996). Integrin- and growthfactor-receptor-induced signaling pathways converge at the
MAP kinases; however, while growth factors cause a transient
activation of the extracellular-signal-regulated kinases 1 and 2
(ERK1/2), the ECM induces a sustained increase of their activity. The latter is associated with increased cyclin D1 expression
(Zhu and Assoian, 1995; Bhmer et al., 1996; Weber et al., 1997).
The ECM also up-regulates cdk inhibitors (CKI) p27kip1 (p27)
and p21cip1/wafl (p21) levels, and this is a mechanism contributing to ECM-induced cell-cycle arrest (Koyama et al., 1996;
Raines et al., 2000).
The composition of the ECM can be responsible for the
recruitment of specific cell types during wound healing. For
example, fibronectin and type I collagen are chemotactic to and

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promote adhesion of most cell types, whereas laminin and type

IV collagen are selective for certain cell types. Tenascin, on the
other hand, is anti-adhesive. Thus, the composition of ECM can
determine which cells are enlisted during wound healing and
whether healing occurs by regeneration or repair.
Many growth factors are sequestered in the ECM, and ECM
interactions can modify the binding of growth factors to their
cell-surface receptors (Schonherr and Hausser, 2000). ECM components can bind to these molecules at multiple sites, and the
proteoglycans can interact through protein core as well as glycosaminoglycan chains. During inflammation, soluble mediators such as growth factors, cytokines, and chemokines are
secreted by inflammatory cells, and some mediators are also
released from damaged tissue by degradation. The activities of
these molecules are directed toward cells participating in
wound healing. On the other hand, the activity of growth factors sequestered in the ECM may be limited to the cell types
needed for the maintenance of normal tissue homeostasis. Some
of the sequestered molecules and ECM components may be tissue-specific in their distribution. The ECM composition and
growth factors available together are likely to regulate which
receptors are expressed and which biochemical signaling events
are induced (Saito and Narayanan, 1999; Cuklerman et al., 2001;
Heldin, 2001). In addition, they will also determine which pathway is chosen among a network of several parallel pathways
and how cells respond functionally; these in turn will determine
the course of wound-healing events.
Other factors that determine cellular response include cell
density and interaction with other cells. The importance of
intercellular interactions is illustrated by the stromal response
to epithelial injury and vice versa, and by the differentiation of
embryonic cells into teratocarcinomas when injected into nude
mice (Bradley, 1990; Denker et al., 1999; Zieske, 2001).
The structural integrity and unique biochemical composition of the ECM are likely to be essential for tissue homeostasis
under healthy conditions and for regeneration after injury. The
importance of local environment in influencing cell behavior is
illustrated by the expression of adult progenitor phenotype
cells by fetal hematopoietic stem cells implanted into the adult
spleen (Geiger et al., 1998). The composition of the ECM
changes with the progression of wound healing. For instance,
during the early stages, when granulation tissue is actively
being constituted, types III, IV, V, and VI collagen, fibronectin,
and vitronectin are actively expressed, and these are replaced
by type I collagen at later stages (Eckes et al., 2000). The changing composition and quality of the ECM do not mimic the local
environment of the healthy tissue. The type and concentration
of polypeptide mediators available change continuously during the course of wound healing. Thus, destruction of the local
environment and failure to reconstitute it are likely to be significant reasons for the failure to regenerate after injury. In this
case, the provisional matrix offered by the granulation tissue
may be conducive for repair rather than regeneration.
It is now recognized that cells needed for regeneration are
present in most tissues. These cells may be "stem cells" removed
from terminal differentiation by several stages, or precursor or
progenitor cells immediately before full differentiation (Fig. 1).
An interesting possibility is that, whereas pre-existing ECM
may provide signals for precursor cells to differentiate in
healthy adult tissues, the ECM synthesized by a "stem" cell and
differentiating stem cell may contribute to the local environment for the differentiation of the next stage cell (Fig. 2).
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Figure 2. Hypothetical model for maturation into fully differentiated

cell. Under healthy conditions, pre-existing healthy matrix recruits
precursor cells that are close to final differentiation and permits their
differentiation to occur. During inflammation and wound repair,
undifferentiated "stem" cells, presumably separated by several stages
from full differentiation, interact with growth factors (GF) and available matrix, differentiate to the next stage, and produce matrix. This
cell interacts with the matrix, which contains the ECM produced by
the previous stage cell, and differentiates to the next stage. This
process continues until the cell becomes fully differentiated.

Local Factors in Periodontal Healing

Many of the events, growth factors, and soluble mediators
described above also participate in the healing of periodontal
tissues after injury by trauma and other causes, including infection. The periodontium has been shown to contain biologically
active mediators, and their role has been recently reviewed
(Cochran and Wozney, 1999; MacNeil and Somerman, 1999;
Bartold et al., 2000; Saygin et al., 2000). The concentrations of
these molecules vary in different periodontal components, and
appear to be relatively higher in alveolar bone and cementum.
The inability of these molecules to be detected in soft tissues,
especially in the gingiva, is likely to be due to their low concentration rather than to their absence (Miki et al., 1987;
Nishimura et al., 1989; Somerman et al., 1989; Nakae et al., 1991).
Although substances present in one periodontal component
can influence other periodontal structures, under healthy conditions these factors are likely to affect mostly the adjacent cells.
Under pathological conditions, the role of these components is
likely to be minor, due to destruction of and biochemical alterations in the local environment and because of the presence of
serum and inflammatory-cell products in relatively large
amounts.

Biology of Cementum
Although cementum is anatomically an integral part of the
tooth, functionally it is a component of the periodontium, and
its major role is to serve as the site of attachment for principal
collagen fibers (Sharpey's fibers). The biology of cementum and
molecular and cellular aspects of cementogenesis have been
discussed elsewhere (Schroeder, 1992; Bosshardt and Selvig,
1997; Saygin et al., 2000), and here we will review only those
aspects relevant to periodontal healing. Three cementum types
differing in the presence of cells and collagen fibers are distinguished in humans. The acellular afibrillar cementum covers
teeth at and along the cemento-enamel junction; it consists of
mineralized matrix, but lacks collagen fibrils and embedded
cells. It is deposited in isolated patches over enamel and dentin,

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and its deposition begins at the end of enamel maturation and

continues for an indefinite time (Bosshardt and Selvig, 1997).
The cells responsible for depositing this cementum have not
been identified, and it appears that connective tissue cells lay
the matrix when they come into contact with the enamel surface. Acellular extrinsic fiber cementum (AEFC), which serves
the primary attachment function, covers cervical and middle
portions of the roots, and it is usually confined to the coronal
half of the root. It consists of a dense fringe of collagenous
fibers implanted into dentinal matrix and, most importantly,
perpendicular to the root surface. The production of AEFC
commences shortly after crown formation and continues to
grow as long as the adjacent periodontal ligament remains
undisturbed. It is produced by cementoblasts that differentiate
closest to the advancing root edge. Cellular intrinsic fiber
cementum (CIFC) is found on dentin where no acellular extrinsic fiber cementum has been laid down, and its formation commences closest to the advancing root edge on the forming root.
This cementum contains cementocytes entrapped in the mineralized matrix, and collagen fibers present in this cementum are
parallel to the root surface. The CIFC has mainly a repair function. The CIFC may overgrow acellular extrinsic fiber cementum and vice versa, and this is called mixed stratified cementum. This cementum is confined to apical root portions and furcations, and it appears to participate mainly in the repair of previously resorbed roots (Schroeder, 1992; Bosshardt and Selvig,
1997).
In rodents, cementogenesis begins with the deposition of a
matrix on the dentin surface by Hertwig's epithelial root sheath
(HERS), disruption of the HERS, migration and organization
by ectomesenchymal cells from the dental papilla, and their
subsequent differentiation into cementoblasts (Cho and
Garant, 1989; Slavkin et al., 1989; Thomas and Kollar, 1989). The
matrix they produce surrounds cells producing cementum, the
cementoblasts, and the cementoblasts are generated by differentiation of progenitor cells, which in mice are believed to arise
from the dental follicle. Epithelial cells of the Hertwig's root
sheath are believed to undergo epithelial mesenchymal transformation (Pitaru et al., 1994; Bosshardt and Selvig, 1997).
The organic matrix of cementum is composed primarily of
collagens. Type I collagen, which plays structural as well as
morphogenic roles and provides scaffolding for mineral crystals, is the major species, and it accounts for 90% of all collagens. The type III collagen, which coats type I collagen fibrils,
makes up ~ 5%. Cementum contains two major non-collagenous proteins, bone sialoprotein (BSP) and osteopontin (OPN).
These proteins, which are prominently expressed in acellular
extrinsic fiber cementum and acellular afibrillar cementum,
remain bound to the collagen matrix, and they possess cell
attachment properties through the arg-gly-asp (RGD) sequence
(Ganss et al., 1999; Sodek et al., 2000). Both proteins are
expressed during early tooth root development by cells along
the root surface. Root-surface cells express the BSP, and it is also
present in mature teeth. In contrast, OPN is present within the
periodontal ligament region of mature teeth. These two proteins are believed to play a major role in the differentiation of
the cementoblast progenitor cells to cementoblasts (Saygin et
al., 2000). The BSP is believed to serve adhesion function for
root-surface cells and to participate in initiating mineralization.
It is chemotactic to pre-cementoblasts and promotes their
adhesion and differentiation (Somerman et al., 1990; MacNeil et
al., 1994). The OPN is expressed by many cells during periods

478

of cementogenic activity. It regulates cell migration, differentiation, and survival via interactions with avb3 integrin, and it
participates in inflammation by regulating monocytemacrophage activation, phagocytosis, and nitric oxide production (Giachelli and Steitz, 2000). In teeth and cementum, it may
regulate biomineralization by at least two mechanisms: regulating bone cell differentiation, and matrix mineralization.
Fibronectin, which is believed to bind cells to the ECM, and
tenascin are present in the basement membrane of HERS during odontoblast differentiation, and later at the attachment site
of periodontal ligament to the root surface. Other matrix components found in the cementum matrix include osteonectin,
which is expressed by cementoblasts producing cellular extrinsic fiber cementum and cellular intrinsic fiber cementum,
osteocalcin, and laminin. Several polypeptide growth factors
with ability to promote the proliferation and differentiation of
putative cementoblasts are sequestered in the cementum
matrix. These include BMP-2, -3, and 4, PDGF, a- and bFGFs,
TGF-b, and insulin-like growth factor-I (IGF-I) (Table)
(Cochran and Wozney, 1999; MacNeil and Somerman, 1999;
Saygin et al., 2000).
Many of these components are also present in bone; however, molecules unique to cementum have also been described.
One of these is an IGF-I isoform referred to as cementum
growth factor (CGF). This is a 14-kDa protein, which is larger
than IGF-I in molecular size (Ikezawa et al., 1997). The second
molecule is a collagenous protein referred to as cementum
attachment protein (CAP). Antibodies to CAP immunostain
only cementum and not other periodontal components or other
tissues (Arzate et al., 1992). In bovine tooth germs, the CAP is
expressed by cementoblasts, and in cementum its expression
pattern is different from that of type I collagen (Saito et al.,
2001). The CAP promotes the adhesion and spreading of mesenchymal cells; however, it promotes the adhesion of mineralized-tissue-forming cells preferentially (Olson et al., 1991;
Pitaru et al., 1995).
The structure and biochemical composition of cementum
are affected by several diseases (Polson, 1986). In periodontitis,
the chronic inflammatory process destroys collagen fibers of
the gingiva, and this may extend to the root surface. In this disease, one significant pathological change is deposition of bacterial plaque substances, including the bacterial endotoxins.
Destruction of connective tissue fibers leads to attachment loss,
and damage to cementum becomes irreversible when the
cementum surface is exposed to periodontal pockets.
Alteration in the biochemical composition of cementum during
periodontal disease results in loss of active substances and
deposition of inhibitors such as endotoxins. Diseased cementum inhibits connective tissue cell attachment and growth and
promotes epithelial attachment (Terranova and Martin, 1982;
Polson, 1986); this was the rationale for new therapeutic
approaches in which diseased roots are conditioned to promote
connective tissue attachment (Bartold et al., 2000).
It is perhaps worthwhile to emphasize that certain aspects
of cementogenesis and cementum biology (such as attachment
mode of cementum to dentin, the rate of cementum apposition)
seem to differ between species, most notably between rodents
and large mammals, including humans (Schroeder, 1992;
Bosshardt and Schroeder, 1996). It still remains unclear whether
these differences are due to spatio-temporal particularities
(tooth size, the pace of development, etc.) or whether they mirror different molecular and/or cellular aspects of cementum

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formation and maintenance. Most of the information available

is descriptive, and it is particularly scant with respect to
humans. Resolving these issues is important, and, at this time,
data derived from animal models must be used with great caution before conclusions for human applications are drawn. The
in vitro/in vivo animal and human models developed recently
(and described in more detail in the last sections of this review)
should be excellent systems to clarify the interspecies differences at the cellular and molecular levels.

Possible Role of Cementum

in Periodontal Regeneration
One major goal of regenerative periodontal therapy is new
Figure 3. Cell activities required for new cementum and attachment
formation and cementum components that possibly regulate these
cementum formation and restoration of soft-tissue attachment
processes. The list of molecules is not complete, and only some examto the cementum. Cementum regeneration requires cementoples are given.
blasts, and the origin of cementoblasts and the molecular factors regulating their recruitment and differentiation are not
fully understood. In vivo animal models to evaluate cementoifest cell specificity. Cell specificity can be achieved in several
genesis during tooth development, the expression pattern of
waysfor example, growth factors targeting specific cell types,
specific matrix molecules, and in vitro experiments studying
unique ECM composition, and conditions permissive for needthe effects of cementum components on periodontal cells have
ed cells but refractory to other cells. Evidence indicates that
provided important clues on how cementum components can
cementum components can regulate cellular activities by all of
regulate cementum regeneration (McCulloch, 1993; Thesleff
these mechanisms. For example, cementum contains molecules
and Nieminen, 1996; Ten Cate, 1997; MacNeil and Somerman,
that promote chemotactic migration, adhesion, proliferation,
1999; Saygin et al., 2000). During tooth development, dental foland differentiation of some periodontal cell types better than
licle cells of ectomesenchymal origin appear to be responsible
others, and these molecules are not detectable in other periofor cementogenesis. In adult mice, the cementoblasts are
dontal structures (Nishimura et al., 1989; Somerman et al., 1989;
believed to be recruited as progenitor cells located paravascuPitaru et al., 1995; Arzate et al., 1996; Ikezawa et al., 1997;
larly in the periodontal ligament or in endosteal spaces of the
BarKana et al., 2000). Adhesion molecules that cause negative
alveolar bone (McCulloch, 1993; Pitaru et al., 1994). Although
selection by excluding unwanted cells are also present in the
cementum formation in rodents differs from that in mammals
cementum (Olson et al., 1991). This cell specificity is not mani(Bosshardt and Schroeder, 1996), the observation of Liu et al.
fested by other ubiquitous matrix molecules, such as fibro(1997) indicates that the periodontal ligament may be one
nectin. Most significantly, the cementum microenvironment
source of cementoblast progenitors in adult humans. These
contains all the components necessary for cell recruitment, proinvestigators demonstrated that a small proportion of clones of
cells cultured from human periodontal ligament form cementum-like
TABLE
mineralized nodules in culture and
Some Important Molecules Identified in Cementum and Their Activity
produce cementum-specific markers
(Bar-Kana et al., 1998). The cementoMolecule
Biological Activity
blasts may also be derived from stem
cells present in the periodontal ligaGrowth factors
ment, gingiva, or alveolar bone; this
IGF-1
proliferation, differentiation, matrix synthesis
may be the case in healing periodonFGF
proliferation, differentiation, matrix synthesis, angiogenesis
tium, when the pool of available
PDGF
proliferation, differentiation, matrix synthesis
progenitors is likely to be reduced or
TGF-
matrix synthesis, angiogenesis, chemotaxis
absent. However, the molecules
BMPs
matrix synthesis, differentiation, bone formation
responsible for recruiting these cells
EGF
proliferation, differentiation
and for their differentiation have not
CGFa
proliferation, differentiationb
been identified. The mechanisms
involved are likely to be more comMatrix components
plex.
A variety of chemotactic factors,
Collagens
cell adhesion, differentiation; regulates proliferation
adhesion molecules, growth factors,
BSP
cell adhesion, differentiation, mineralization
and ECM constituents participate
OPN
cell adhesion; regulates differentiation and survival
together in the recruitment of cemenFibronectin
cell adhesion, differentiation, regulates proliferation
toblast progenitors, their expansion,
Osteonectin
regulates angiogenesis, differentiation, and proliferation
and differentiation (Table, Fig. 3).
Cementumcell adhesion, differentiationb
Many of the same components may
attachment protein
be available during periodontal healing; however, most of these molea Cementum-derived growth factor, isoform of IGF-1.
cules are pleiotropic and do not manb Mineralized tissue-forming cells respond better than fibroblasts to these proteins.
13(6):474-484 (2002)

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479

liferation, and differentiation, and molecules from the circulation are not necessary (Table, Fig. 3). For example, cells can
escape cell cycle arrest and complete cell division and differentiation in the presence of cementum proteins alone (Yokokoji
and Narayanan, 2001). Needed molecules may also be present
in enamel matrix extracts, although in this case the active components may or may not be the same as those of the cementum
matrix.
The mechanism by which selection of cementoblast progenitors is achieved is unclear, and it most likely involves specific integrins and signaling events (Liu et al., 1997; Ivanovski
et al., 1999; Saito and Narayanan, 1999; Komaki et al., 2000).
Once selected, the progenitor cells adhere to the root surface,
and their expansion could be facilitated by growth factors present in the cementum matrix. Growth factors identified in
cementum include IGF-I, FGFs, EGF, BMPs, and TGF-b
(MacNeil and Somerman, 1999). Although selection of cells
can be achieved at the level of adhesion, it could also be by
preferential proliferation. For example, the CGF is an isoform
of IGF-I, and fibroblasts mitogenically respond to both growth
factors similarly; however, osteoblasts respond better to the
CGF (Ikezawa et al., 1997).
These observations underscore the importance of restoring
or providing the cementum microenvironment to initiate and
promote new cementum formation. The integrity of cementum
is chemically altered by disease due to deposition of bacterial
endotoxins, and diseased cementum is removed during therapy. Dentin not covered by cementum undergoes resorption.
Root conditioning is not likely to restore the original composition of the cementum local environment, and instead exposes
molecules, especially type I collagen, which manifest poor cell
specificity (Polson, 1986). Applying some growth factors is not
likely to provide the complete repertoire of the needed molecules, the concentration and type of which change continuously during the healing process. Similarly, while barrier membranes can facilitate population of the site by needed cells, they
are not likely to provide the local environment necessary for
their differentiation. Further, providing enamel proteins
(Lindhe, 1997; Hirooka, 1998), while likely to be conducive to
early cementogenesis, may not provide appropriate environment for recruiting cementoblast progenitors in adults and for
their differentiation. ECM components are indeed expressed
during periodontal healing (Lekic et al., 1996; Ivanovski et al.,
2000); however, whether all molecules present in cementum
matrix are expressed is not known. The temporal sequence of
their expression is also important for initiating new cementum
formation. These factors may explain why cementum regeneration is not always predictable for the available regenerative
procedures.
During inflammation and wound-healing response following periodontal surgery, a battery of growth factors is available
from both the circulatory and inflammatory cells. The provisional matrix formed by blood clot and granulation tissue has
a composition different from that of the cementum environment under healthy conditions. This combination of growth
factors and ECM is not likely to be conducive to the selection
and function of cementoblast progenitors. If progenitors are not
present and only stem cells are available, their differentiation to
cementoblasts may require that events during early cementogenesis be recapitulated, and needed signaling molecules are
not likely to be available in the wound-healing environment.
Thus, it appears essential that the right combination of ECM

480

components and growth factors is necessary to induce new

cementum formation.
One factor that needs to be considered is that, in early and
moderate periodontitis, acellular cementum (coronal half of the
root) is affected, and the damage extends to cellular cementum
in most advanced and furcally positioned lesions. These surfaces are almost always covered by cellular cementum during
successful regeneration; whether this is adequate is unclear
(MacNeil and Somerman, 1999).
The growth factors and adhesion molecules present in
cementum are also active toward cells of the gingiva, periodontal ligament, and alveolar bone (Narayanan and Bartold,
1996; Bartold et al., 2000). Therefore, it is possible that cementum components have the potential to participate in the regulation of homeostasis and regeneration of these tissues.
However, this is perhaps not significant under healthy conditions, because the growth factors present in cementum remain
bound to the cementum matrix. Even if the inflammatory
process releases them, their relative concentrations are likely to
be less than those available from the blood and inflammatory
cells; therefore, contributions by cementum molecules to the
regeneration of other periodontal tissues are likely to be marginal or insignificant.

Models of Cementoblastic Cells and Their

Potential Application for Developing New
Strategies for Periodontal Regeneration
In this section, we will focus on an emerging approach to the
development of new treatment modalities for restoration of
attachment that involve possible application of cementoblastic cells.
Viable cementoblasts and/or periodontal ligament cells
near the cementum appear to play a critical role in the regeneration of the tooth attachment apparatus. It is a well-known fact
in the clinic that if an avulsed tooth is replanted into the tooth
socket shortly after the avulsion (or the tooth is stored in the
conditions that allow for cell survival), cementum-mediated
attachment is efficiently re-established. In contrast, if the
avulsed tooth is replanted without viable cells present, the
healing process is frequently impaired, and severe complications (i.e., ankylosis, root resorption) are more likely to develop
(Boyd et al., 2000). This indicates that viable cementoblasts
and/or intact molecules associated with them, in addition to
cementum matrix (discussed above), are likely to be actively
involved in recruiting cells that next differentiate into cementoblasts and form new cementum that is critical for re-establishing structurally and functionally sound attachment. Thus, the
strategy of investigating cementoblastic cells as a possible
source of factors that are specifically involved in cementum
regeneration/healing has a clear basis in a well-known clinical
observation.
In recent years, several laboratories have reported successful isolation and propagation of cells (from both animal and
human sources) exhibiting an apparent cementoblastic phenotype in vitro (D'Errico et al., 1995, 1997; Grzesik et al., 1998).
These cells have been shown, reproducibly, to form histologically proven cementum-like tissue following in vivo transplantation into immunodeficient mice (Grzesik et al., 1998, 2000;
Handa et al., 2002). One of the most important benefits of the
establishment of such combined in vitro/in vivo models is that
they will allow for elucidation of the relationship between

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osteoblasts and cementoblasts. In contrast to bone, very little is

known regarding the specific mechanisms involved in the
maintenance of cementum structure and function in humans
on the cellular and molecular level. Most of the information
available is an extension of what is known for bone, and the
general assumption is that cementoblasts' physiology closely
follows that of osteoblasts. This may well be true in a broad
sense; however, given the clear differences in the structure and
function between these two tissues, it is conceivable that different factor(s) are directing the distinct aspects of differentiation
of osteoblasts and cementoblasts, respectively. This paradigm
is of particular significance for the development of strategies
for regenerating cementum with cell-based therapies (see
below). Bone, in contrast to cementum, undergoes constant
remodeling carried out by osteoclasts and is accompanied by
bone marrow. The deposition of bone on the root surface by
osteoblasts or osteoblastic precursors (either delivered or targeted to the tooth root surface) is therefore likely to result in the
recruitment of osteoclasts, followed by the development of
bone marrow; this, in turn, would significantly alter the local
environment in periodontium and may result in root resorption, ankylosis, and subsequent loss of attachment. Thus, novel
in vitro/in vivo models offer promise toward developing relatively simple and faithful means of recruiting cells with cementoblastic potential and their differentiation and excluding
unwanted osteoblastic precursors. Last, the use of these systems allows for direct in vivo experimentation using human
cells. This may be particularly important, because cementum
has been reported to differ significantly between species in
some aspects of its physiology (Schroeder, 1992; Bosshardt and
Schroeder, 1996).
The fact that cells with a cementoblastic phenotype can be
cultured also offers the possibility for development of a more
radical treatment of cases where most of the cementum in situ
as well as adjacent soft tissues has been lost due to pathology.
In such a scenario, re-establishing the proper microenvironment (discussed elsewhere) will not be sufficient to achieve
regeneration, due to the lack (or low abundance) of available
precursors/progenitors of cementoblasts in the affected area.
In principle, cementogenic cells can be isolated from a relatively small specimen, expanded in culture, and subsequently retransplanted to the same patient. Primary human cementoblasts can be efficiently grown from dissected fragments of
healthy tooth root cementum treated with collagenase. The culture conditions are fairly standardit is sufficient to maintain
these cells in standard tissue culture medium (alpha minimal
essential medium [MEM], Dulbecco's MEM/F12) supplemented with 10% fetal bovine serum (Grzesik et al., 1998, 2000). In
this manner, it is feasible to obtain large numbers (in the range
of 108-109) of committed cementogenic cells from a single tooth.
However, this system has a significant drawback because of the
requirement that healthy teeth be extracted for the cultures to
be established. Also, it is still not clearly established whether
cementogenic cells can be reproducibly obtained, by this
approach, from teeth of aged and/or diseased patients. These
reservations somewhat deter the enthusiasm for the cell-transplantation approach. Such a scenario would be undoubtedly
more feasible if cells with cementogenic potential (or cells that
can be induced toward cementoblastic differentiation) could be
obtained from sources other than cementum, such as soft periodontal tissues (periodontal ligament, gingiva) and/or even
remote sites (bone marrow, fat). The use of sources other than
13(6):474-484 (2002)

cementum for cell isolation, however, may require two critical

conditions:
(1) Exclusion of unwanted cell lineages; this can be accomplished by the selection of specific cells before culturing,
selection during culture expansion, and/or selection
prior to delivery to the defect. Although molecules such
as CAP show some promise (Liu et al., 1997; BarKana et
al., 2000), currently, no defined molecules are known to
discriminate between cementogenic and non-cementogenic but osteogenic precursors.
(2) Inducing differentiation along the cementoblastic lineagethis can be accomplished by pre-treatment of cells
in vitro or in vivo (i.e., the application of an inducing stimulus concomitantly with cell delivery to the patient), or
both, with a specific inducing factor. At this time, however, no defined molecular factor specifically inducing the
cementoblastic phenotype is known.
Obviously, at this time, the application of cell therapy for
treating cementum loss using cells isolated from soft tissues
remains a highly hypothetical option, because methodology to
satisfy these two conditions is not available. The first steps
would be, then, to solve these two major problems relating to
selecting cells with a proper differentiation potential and to test
the general feasibility of this complex approach. Clearly, the
recently developed in vitro/in vivo combined models of cementogenesis will be of great use for these purposes. In principle,
the effect of any factor and its relation to the cementoblastic
phenotype can be meaningfully studied in both the in vitro and
in vivo conditions. It cannot be excluded that some of the
already-known molecules, including pharmacological agents,
are indeed capable of inducing cementoblastic differentiationfor example, a well-known immunosuppressant,
cyclosporin A, has been shown to induce development of
cementum in the gingival tissue of rats (Ayanoglou, 1998).
Similarly, the in vitro/in vivo system can also be used for further
elucidation of the modes of action of some of the developed
and currently tested compounds (such as Emdogain) with
apparent cementum-/growth-promoting activities.
Once the molecules critical for cementum regeneration are
identified, an efficient and relatively simple delivery system
must be developed if they are to be used for periodontal regeneration. One of the major problems of applying bioactive molecules in in vivo settings is that they need to be present in a specific location and remain bio-available and bioactive for
extended periods of time. For cementum regeneration, its
anatomyi.e., the fact that cementum is a mineralized "interface" tissue connecting mineralized dentin and non-mineralized fibrous periodontal ligamentimplies that a successful
delivery system should somehow reconcile these two distinct
environments. An additional complication is that regenerated
cementum must be strictly confined only to the tooth root surface, without compromising the integrity and structure of the
periodontal ligament. One approach would be to target and
bind bioactive molecules to the mineralized surface of the tooth
root. Although some of the growth/differentiation factors, such
as BMPs, have affinity to mineral of bones and teeth, many do
not display this preference. The lack of efficient and stable
binding of biologically active molecules to mineralized tooth
root surfaces is likely to account for, at best, moderate and
unpredictable therapeutic effects of many of those substances
tested so far (Caffesse et al., 1991; Choi et al., 1993).
In the last decade, several proteins have been found to

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481

bind mineral with high affinity, and this activity has been firmly linked to acidic motifs (poly-aspartate and poly-glutamate
"stretches") present in these molecules. It is possible to utilize
fusion proteins consisting of a bioactive part(s) (corresponding
to factor[s] promoting cementoblastic differentiation, such as
the CAP), and the mineral-binding domain derived from a different protein could allow for efficient targeting to the tooth
root surface.
Integrin-matrix interactions are fundamental to most
aspects of cellular metabolism and are especially important for
the healing/regeneration and maintenance of the structure and
proper functions of tissues and organs. Importantly, there is a
clear positive correlation between integrin ligation and cell survival (Meredith et al., 1996). For most integrin ligands, the
receptor-binding domains have been mapped, and synthetic
peptides, corresponding to these sequences, are biologically
active. Several integrins as well as their natural ligands (e.g.,
collagens, BSP, OPN, CAP) are known to be expressed by
cementoblastic cells (Steffensen et al., 1992; Ivanovski et al.,
2000), implying that they may be important regulators of
cementogenesis. This also indicates that targeting integrin
receptors may be beneficial for stimulating cementum regeneration. For example, in our laboratory, we have recently established that synthetic peptides containing the integrin-binding
RGD sequence and the polyglutamate domain derived from
BSP can be efficiently bound to synthetic hydroxyapatite/tricalcium phosphate ceramic. When such ceramics were next
used as a vehicle for transplanting cementogenic cells into
immunodeficient mice, cementum formed more efficiently and
abundantly than when unaltered ceramics were used (Grzesik
et al., unpublished data). This result indicates that integrins
may be directly involved in some aspects of cementum formation and that increasing the numbers of active adhesive integrin ligands on mineralized surfaces enhances formation of
cementum by committed cementogenic cells. Currently, we are
evaluating whether this approach can be utilized for directing
bioactive peptides to naturally occurring mineral matrices of
bone and dentin in an experimental setting.
In summary, these are still early days of periodontal tissue
regeneration via cell- and matrix-based therapies. Nevertheless,
recent developments in basic science indicate that these
approaches are undoubtedly feasible and, given their promise,
worth exploring.

Acknowledgments
This work was supported by NIH grants DE-13061, DE-08229, DE10491, DE-13475, and DE 14016.

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