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Periodontology 2000, Vol.

24, 2000, 7398 Copyright C Munksgaard 2000


Printed in Denmark All rights reserved
PERIODONTOLOGY 2000
ISSN 0906-6713
Molecular and cell biology of
cementum
NAZAN E. SAYGIN, WILLIAM V. GIANNOBILE & MARTHA J. SOMERMAN
Cementum was rst described in 1835 (53), and yet
until recently has remained a poorly dened tissue
at the cellular and molecular level. While research
through the years has demonstrated that cementum
is a unique tissue histologically (19, 21), examination
of the proteins expressed by cells forming ce-
mentum, cementoblasts has not resulted in deni-
tive identication of proteins specic to cementum
(141). In fact, unlike dentin and enamel, where there
are clear differences in the proteins present in these
tissues and the factors regulating their function
when compared with bone, cementum appears to
contain factors in common with those associated
with bone and to be developmentally controlled by
similar factors. Alternatively, it has been suggested
that factors supplied by epithelial cells within the lo-
cal environment may, at least in part, inuence the
differentiation of follicle cells into cementoblasts,
thereby laying down cementum (67), or in fact that
epithelial root sheath cells undergo transformation
into cementoblasts and thus provide the appropriate
matrix for cementum formation (Fig. 1) (18, 21).
Thus, the dogma that follicle cells have the capacity
to differentiate into an osteoblast, a cementoblast or
a periodontal ligament broblast has never been
proven and is now being challenged. This chapter
will attempt to present existing knowledge as to the
cellular and molecular properties of cementum in a
framework that provides the clinician the infor-
mation necessary for careful evaluation of agents
that are being marketed for their ability to regenerate
periodontal tissues, with a specic focus on agents
targeted at regeneration of cementum.
There is accumulating histological evidence that
cementum formation is critical for appropriate
maturation of the periodontium, both during devel-
opment as well as that associated with regeneration
of periodontal tissues. Despite many years of re-
search and the importance that cementum is
thought to play in the reparative process following
periodontal disease, very little is known about the
73
cells responsible for formation of cementum, ce-
mentoblasts. The wealth of what is known about ce-
mentum comes from numerous, detailed studies of
its histology and composition, which will be touched
on only briey here since the focus of this chapter is
on the cellular and molecular properties of this
tissue (1922, 199). Light and electron microscopy
has enabled cementum to be classied into ve dif-
ferent subtypes based on the presence (cellular) or
absence (acellular) of cells and the source of collagen
bers (extrinsic versus intrinsic). All of these sub-
types are quite different from bone, in that they are
not innervated, exhibit little or no remodeling and
are avascular. Despite these differences, cementum
is very similar to bone. First, diseases that affect the
properties of bone, often alter cementums prop-
erties as well. For example, Pagets disease results in
hypercementosis, hypophosphatasia results in no
cementum formation, with exfoliation of teeth, de-
creased cementum is associated with hypopituitar-
ism and defective cementum is seen in patients with
cleidocranial dysplasia (175). Second, the compo-
sition of cementum is similar to that of bone. Ce-
mentum is approximately 50% hydroxyapatite and
50% collagen and noncollagenous proteins. Protein
extracts of mature cementum promote cell attach-
ment, migration and stimulate protein synthesis of
gingival broblasts and periodontal ligament cells.
Investigation of these extracts revealed the presence
of bone sialoprotein, osteopontin, vitronectin and
bronectin. Immunocytochemistry and in situ hy-
bridization conrmed the presence of these proteins
and further identied osteocalcin, g-carboxyglutam-
ic acid, osteonectin, proteoglycans and several
growth factors. Two additional molecules, an ad-
hesion molecule and a growth factor, have been
identied and initial data suggest that they may be
unique to cementum. Cementum attachment pro-
tein may prove to be a cementum-specic collagen-
like molecule, while a factor initially named ce-
mentum-derived growth factor, now considered to
Saygin et al.
be an insulin-like growth factor-Ilike molecule may
prove to have properties similar to those of insulin-
like growth factor-I (see Table 1 for references).
In this chapter, the factors above are considered
in terms of their potential for having an active role
in triggering cementum formation, contrasting the
role of specic factors during development of ce-
mentum versus repair and regeneration of ce-
mentum. Also, existing evidence as to the success of
such agents in regeneration of cementum in vivo will
be discussed. The next section is devoted to a dis-
cussion on models used to investigate the properties
of cementum, while the nal section is more specu-
lative in nature, providing a discussion on the im-
pact of information obtained from ongoing studies
at the cellular and molecular level and at the tissue
engineering level, to the design of attractive therap-
ies for regeneration of periodontal tissues.
Regulators of cementogenesis
Overview: events, cells and factors
associated with cementum
Events
As shown in Fig. 1 and 2, while many of the events
required for formation of cementum are well estab-
lished, the actual cells and factors required to form
this tissue during development as well as during re-
generation have yet to be dened. Ideal agents to
use in attempts to regenerate tissues would include
those having the ability to promote migration and
attachment of appropriate cells to the healing site
with subsequent orchestration of cells to allow for
cell differentiation, that is, to act as an osteoblast,
cementoblast or periodontal ligament broblast.
Equally important would be the ability of an agent
to promote mineralization (new cementum) along
the root surface, with insertion of periodontal liga-
ment into cementum and opposing alveolar bone to
form the periodontium. This simplied description
of events suggests that regeneration recapitulates
development (Fig. 2). However, it is important to rec-
ognize that there are differences in both events and
cells required for these two processes. For example,
proper clot formation appears to be critical for ade-
quate wound healing and subsequent regeneration
of any tissue. In addition, during wound healing
there is a normal inammatory response, resulting
in release of several cytokines and growth factors
that may not be associated with development of a
given tissue. In spite of these differences, it is poss-
74
ible that factors identied as having a role in regulat-
ing the developing tooth root cementum, but not
necessarily associated with regeneration of this
tissue, may prove of value for use in regenerative
therapies. As depicted in Table 1 and discussed be-
low, while many of these factors are known to be
present during development and regeneration of
periodontal tissues, their precise functions in these
tissues are not clearly dened. Ongoing research tar-
geted at dening the importance of these factors to
cell function will help to clarify the role of these fac-
tors in regulating periodontal tissues.
Cells
Histological examination of the healthy periodon-
tium indicates that several types of mesenchymal
cells are important for maintenance of a healthy
periodontium. Such cells include: periodontal liga-
ment broblasts, responsible for ensuring a func-
tional periodontal ligament region; osteoblasts and
associated progenitor cells, responsible for preserv-
ing the surrounding alveolar bone; cementoblasts,
root surface lining cells, that appear to be limited in
function in health but may be activated during
wound healing, and paravascular/marrow cells, that
are important for providing the required local nutri-
ents at the site. In contrast, the cells responsible for
formation of periodontal tissues, both developmen-
tal and repair/regenerative aspects, are less dened
and an area of intense investigation by several lab-
oratories (Fig. 2).
At the developmental level there is existing evi-
dence suggesting that ectomesenchymal cells, fol-
licle cells and dental papilla cells, when triggered ap-
propriately, have the capacity to act as cemento-
blasts, or periodontal ligament broblasts or
osteoblasts (4, 43, 172, 176, 226), (Fig. 1). In addition,
although it is not the focus of this chapter it is im-
portant to point out the strong evidence for the re-
quirement of follicle cells and factors secreted by
these cells for normal tooth eruption, regardless of
root formation (246, 248). Some of the factors that
may trigger differentiation of follicle cells or possibly
transformation of Hertwigs epithelial rooth sheath
cells so as to function as cementoblasts are dis-
cussed below and listed in Fig. 1 and Table 1. The
specic cell type(s) having the capacity to function
as cementoblasts during wound healing remain(s)
unknown as well. There is some suggestion that a
small population of periodontal ligament cells in the
mature periodontium have the capacity to undergo
differentiation toward an osteoblast or cementoblast
Molecular and cell biology of cementum
Fig. 1. Cementoblast maturation. Top. Depicts possible the intensity of expression). Abbreviations: OB: osteoblast;
cell types involved in developing periodontal tissues. Bot- PDL: periodontal ligament cell; CM: cementoblast; EM:
tom. Genes that are known to be expressed by cells at epithelial-mesenchymal; PTHrP: parathyroid hormone
various stages. Future research targeted at determining related protein; BMP: bone morphogenetic protein; Col.:
the signicance of these factors, as well as yet to be iden- collagen; CSF: colony-stimulating factor; EGF: epidermal
tied factors, to cell activities will assist in the design of growth factor; ALKP: alkaline phosphatase; BSP: bone sia-
predictable regenerative therapies. or indicates loprotein; OC: osteocalcin; OPN: osteopontin.
whether a factor is present or not (but does not indicate
phenotype and thereby act to lay down bone or ce-
mentum (128, 167, 188). However, accumulating evi-
dence exists to a support a role for periodontal liga-
ment broblasts as inhibitors of mineralization (82,
156, 169, 194). Thus, there may be distinct cell popu-
lations within the periodontal ligament region that
can both promote and inhibit mineral tissue forma-
tion depending upon trigger factors. Also, it is highly
likely that other sources of cementobloast or osteo-
blast progenitor cells include marrow stroma and
paravascular and endosteal broblasts (151). At the
research level there is a need to dene both the cells
involved in regeneration as well as the trigger factors
for controlling cell activities, in order to design pre-
dictable regenerative therapies. Discussed below are
specic factors associated with periodontal tissues
during development and in mature and regenerating
75
tissues that may prove to have potential as trigger
factors for use in regenerative therapies targeted at
promoting cementum formation.
Factors
Discussed below and outlined in Table 1 are factors
known to be associated with cementum either dur-
ing development and/or maturation and/or re-
generation. Also included are molecules that have
not been fully characterized but have been reported
to be linked with cementum. Many of these factors
have been implicated as having a role in controlling
several cell activities and thus can be listed under
each activity in Table 1; however, for simplicity, fac-
tors are listed only under the activities currently con-
sidered to be their major function.
Saygin et al.
Fig. 2. Regeneration versus development: events and cells. types involved and in some of the factors promoting cell
While similar events occur during development and re- activities. ERS: Hertwigs epithelial root sheath; PDL: peri-
generation of tissues, there are clear differences in cell odontal ligament.
Adhesion and chemotactic factors
Central to the interaction of cells with the local en-
vironment is the ability of cells, through receptor-
ligand interactions, to be attracted to and remain at
the site of action. Several ligands have been iden-
tied that appear to have an important role in at-
tracting cells to specic sites during tissue develop-
ment. Fibronectin is one of the most extensively
studied of such molecules that, in addition to its role
in tissue development, also is purported to have an
notable role in attracting and maintaining appropri-
ate cells at healing sites. There are several compre-
hensive, excellent reviews that present the import-
ance of adhesion molecules and associated receptors
to a variety of cell activities, and the reader is re-
ferred to these for in-depth information on these
molecules (71, 130). The discussion here will be
limited to adhesion and chemotactic factors that
have been regarded as having a role during develop-
ment and/or regeneration of cementum.
76
Development
Data from in situ hybridization analyses indicate
that the adhesion molecules osteopontin and bone
sialoprotein are expressed by cells along the root sur-
face, cementoblasts, during early stages of tooth root
development. In contrast, cells expressing collagen
are noted throughout the surrounding soft connec-
tive tissue, follicle cells and periodontal ligament
broblasts (139, 142). Bone sialoprotein messenger
RNA and protein remain localized to the root surface
in the mature tooth, while osteopontin protein is
noted within the periodontal ligament region in the
mature tooth (136, 142, 154, 253). In addition to
these adhesion molecules, laminin has been iden-
tied on the dentin surface at the initiation of ce-
mentum formation, where it has been speculated
that this protein serves a role in attracting appropri-
ate cementoblast-like cells to the root surface (145,
146, 235). Further known factors, as well as yet to be
identied novel factors, secreted by epithelial cells
may promote migration and/or adhesion of appro-
Molecular and cell biology of cementum
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77
Saygin et al.
priate cells to the root surface (19, 21, 67, 101, 121).
Importantly, the ratonale behind the use of enamel
matrix derivative (129: see entire issue) is that en-
amel matrix proteins may promote cementoblast ac-
tivity including proliferation, migration/adhesion, as
well as cell differentiation. With regard to cell differ-
entiation this would be analogous to the known abil-
ity of ameloblasts-odontoblasts to secrete products
required for the development of both dentin and en-
amel (epithelial-mesenchymal interactions).
Suggested roles for bone sialoprotein include
acting as an adhesion molecule to maintain appli-
cable cells at the root surface and as an initiator of
mineral formation along the root surface (102, 104,
170, 213, 215, 221). Importantly, the temporal and
spatial expression of bone sialoprotein during ce-
mentogenesis and bone formation is consistent with
a role for this molecule in promoting mineral forma-
tion (16, 3639, 56, 73, 137, 140, 142, 153). Like bone
sialoprotein, the phosphoglycoprotein osteopontin
contains the well recognized adhesion domain, argi-
nine-glycine-aspartic acid (RGD) targeted to specic
integrin receptors, as well as other adhesion regions
that act to promote migration and cell adhesion in
vitro (52, 213, 241, 250, 251). However, unlike bone
sialoprotein, which is selective to mineralized
tissues, osteopontin is expressed by several cells and
localized to several tissues where its function may
relate to post-translational modications within spe-
cic tissues. With regard to bone and cementum, os-
teopontin is expressed during periods of osteogenic/
cementogenic activity in situ (29, 31, 3638, 56, 137,
138, 142, 152, 154, 155, 224), and also is associated
with resorptive activity (58, 147, 182, 203, 224). Oste-
opontin has been linked to regulation of ectopic
crystal formation, where evidence supports a role for
osteopontin in controlling the extent of hydroxyapa-
tite crystal nucleation and/or growth (17, 76, 83,
102104, 204, 209, 219). Also, osteopontin has been
reported to inhibit apoptotic events such as those
associated with inammation (51, 65), and this abil-
ity may have some signicance in the regulation of
cells at sites during development of cementum and
also during wound healing. Thus, while both bone
sialoprotein and osteopontin may have a role in re-
cruitment and maintenance of selective cells at the
root surface, an equally important role may be re-
lated to control of mineralization along the root sur-
face (see below under mineralization).
Type I collagen, the most abundant protein of ce-
mentum, is known to promote cell attachment, but
also is a critical molecule for maintaining the integ-
rity of both soft and hard connective tissues, during
78
development as well as in repair. Less clear is the
role of type XII collagen in developing and in main-
taining a functional periodontal ligament. Studies by
Karimbux (116) and by MacNeil et al. (139) demon-
strated that type XII collagen is expressed by peri-
odontal ligament cells during later stages of root for-
mation but not by follicle cells prior to root develop-
ment. Thus, while it is not clear whether this
collagen has a role in promoting cell adhesion, it
may act through association with type I collagen in
formation and maintenance of a functional peri-
odontal ligament, thus preventing ankylosis.
Regeneration
More recent in vivo studies, using rat periodontal de-
fect models, have shown that both bone sialoprotein
and osteopontin are expressed by cells linked to for-
mation of mineralized tissues, while osteopontin
also is expressed by cells within the newly forming
periodontal ligament (125). Future studies directed
at overexpressing or blocking expression of these
molecules during periodontal wound healing should
provide additional information required to establish
the function of these molecules in mineralized
tissues and also determine the value of using such
agents clinically for enhancing regeneration of peri-
odontal tissues.
Maturation
Mature cementum contains the adhesion molecules
mentioned above as well as vitronectin and ce-
mentum attachment protein. Data to date indicate
that cementum attachment protein shares signi-
cant homology to type I collagen (249). The actual
specicity of cementum attachment protein to ce-
mentum, or in fact whether this is a unique protein,
awaits further research and availability of DNA
probes to determine cells expressing cementum
attachment protein during tooth root development
and maturation (9, 249).
In addition to the classical adhesion molecules
listed in Table 1, proteoglycans, ubiquitous to all
connective tissues, most likely play a role in regulat-
ing cell-cell and cell-matrix interactions both during
development as well as in regeneration of cementum
(11). Proteoglycans are linked to several aspects of
the cell from the matrix, to cell surfaces, to cell or-
ganelles. They are highly anionic molecules impli-
cated in playing a role in several cell functions in-
cluding tissue hydration, storage for growth factors
thereby regulating activity of growth factors, and in
controlling collagen ber formation, cell adhesion,
cell growth and cell differentiation.
Molecular and cell biology of cementum
Mitogens
Details on the potential role of mitogens in control-
ling cell function are provided later. It is clear that
factors that promote cell proliferation offer a critical
mass of cells at a given site, as required for sub-
sequent synthesis and secretion of molecules to
form the extracellular matrix needed for cell differ-
entiation and for mineralization.
As shown in Table 1, mitogens that have been
mapped during tooth root development include the
members of the transforming growth factor-b super-
family, growth hormone, insulin-like growth factor-
I/II and parathyroid hormonerelated protein. Many
more growth factors have been identied during
odontogenesis, and these include broblast growth
factor, platelet-derived growth factor, and growth
hormone (59, 60, 231, 256258), but their relation-
ship to root formation have not been detailed. While
these molecules have been called growth factors,
their role during tooth development does not appear
to be associated with proliferative activity. In fact
some transforming growth factor-bs and parathyroid
hormonerelated protein may have a role in regulat-
ing cell differentiation and subsequently mineraliza-
tion, as discussed in the next section.
Inadditionto the factors listedabove, Narayananet
al. (107, 108) extracted a factor from mature ce-
mentum they called cementum-derived growth fac-
tor. This factor has been shown to enhance prolifer-
ation of gingival broblasts and periodontal ligament
cells (107, 108). Cementum-derived growth factor
bears substantial homology toinsulin-like growthfac-
tor-I, and thus additional research is required to de-
termine the uniqueness of this molecule to specic
tissues. In this regard, studies fromWaters group sug-
gest that both insulin-like growth factor and growth
hormone may have independent roles in promoting
cementogenesis, including perhaps the ability of both
of these molecules to induce expression of bone mor-
phogenetic proteins (47, 126, 257).
Differentiation
Signicant efforts have been placed on determining
factors regulating differentiation of cells during root
development into cells that can function as peri-
odontal ligament cells, cementoblasts and/or osteo-
blasts, with the goal of yielding information that may
prove valuable for developing ideal agents for pro-
moting regeneration of periodontal tissues. While the
factors listed in Table 1 are attractive candidates and
some have shown promise at the clinical level (see
79
later) the exact mechanisms, cells or factors required
for promoting formation of periodontal tissues has
yet to be determined. The discussion below provides
a brief overviewof specic factors. The readers are re-
ferred to the original studies and detailed reviews for
more information in this area (4, 77, 89, 129 see en-
tire issue, 245 see entire issue).
Development
It is well established that epithelial-mesenchymal
signaling through specic molecules is required for
formation of many tissues, including lung, heart,
hair follicles, and tooth crown (enamel and dentin)
(135, 231, 233). In contrast, the exact mechanisms or
factors involved in regulating formation of ce-
mentum remain undened. For example, strong evi-
dence exists to support a role for both bone morpho-
genetic protein 2 and 4 as molecules critical to epi-
thelial-mesenchymal signaling events required for
enamel and dentin formation (15, 239); however,
there is no indication that bone morphogenetic pro-
teins 2, 4 or 7, based on expression pattern, serve as
epithelial-mesenchymal signaling molecules or have
any other role in formation of cementum (1). In con-
trast, bone morphogenetic protein-3 may play a role
in cementum formation since bone morphogenetic
protein-3 transcripts are expressed by follicle cells
surrounding murine rst molars (1). More recently,
Thesleffs laboratory in collaboration with our lab-
oratory, using the murine rst molar model, have
mapped expression of bone morphogenetic proteins
at later stages of root development. Notably, of the
bone morphogenetic proteins examined (2, 3, 4 and
7) only bone morphogenetic protein-3 was ex-
pressed at later stages of root development. Bone
morphogenetic protein-3 was selective to root lining
cells and was not expressed by cells within the peri-
odontal ligament region (unpublished data). While
this is speculative at this point, it is possible that
bone morphogenetic protein-3 plays an important
role in differentiation of follicle cells along the ce-
mentoblast pathway.
In terms of the importance of epithelial products
in inuencing cementoblast phenotype several
studies, including immunohistochemical, in situ
hybridization and recombination studies, provide
data that support a role for products secreted by
the epithelial root sheath in regulating cementum
formation. These factors, some as yet to be iden-
tied, may attract cells to the root surface, such as
laminin (145, 146, 235), or promote differentiation
of follicle cells along the cementoblast pathway,
Saygin et al.
such as sheathlin (also called ameloblastin and
amelin) (67, 101, 121).
Alkaline phosphatase is known to regulate forma-
tion of mineralized tissues, including cementum. Re-
cent studies focused on histological examination of
teeth from alkaline phosphatase knock out mice sug-
gest that alkaline phosphatase may have a more
critical role in controlling formation of acellular ce-
mentum versus cellular cementum (14). This is an
interesting and potential important nding in terms
of efforts directed at regeneration of cementum,
where the cementum formed appears to be cellular
in nature. Thus, factors controlling formation of
cellular cementum may be different than those in-
volved in formation of acellular cementum.
Another molecule that may prove to be important
toward expression of the cementoblast phenotype is
parathyroid hormonerelated protein. There is ac-
cumulating evidence that parathyroid hormonere-
lated protein has a role in regulating early stages of
tooth development and tooth eruption (50, 180).
Rescued parathyroid hormonerelated protein
knockout mice are small in stature, exhibit cranial
chondrodystrophy and a failure of tooth eruption
(180). The role for this molecule in tooth root devel-
opment is less clear. Parathyroid hormonerelated
protein messenger RNA and the associated para-
thyroid hormone/parathyroid hormonerelated pro-
tein type I receptor have been identied within de-
veloping periodontal tissues (13, 174, 180, 228), and
an extract of parathyroid hormone was shown to en-
hance both tooth eruption and orthodontic tooth
movement (50, 180).
Other factors such as insulin-like growth factor
and proteoglycans, present in developing and ma-
ture cementum may serve multiple functions includ-
ing monitoring mineralization and controlling cell
differentiation.
In addition to the factors discussed above, several
studies suggest that the transcription factor, core
binding transcription factor 1/osteoblast-specic
transcription factor 2 is a key activator of osteoblast
differentiation (8, 62, 119, 173). Importantly, during
tooth development in mice, osteoblact-specic tran-
scription factor 2 is expressed by mesenchymal cells,
including follicle cells and cementoblasts, as well as
by ameloblasts (113).
Regeneration
As presented later, the factors discussed above may
prove important for promoting regeneration and
thus it is not surprising that some of these agents
have been used in periodontal regenerative models.
80
Platelet-derived growth factor and platelet-derived
growth factor-receptor have been localized to peri-
odontal tissues during wound healing (85). In this
regard, an apparent limitation to current regenera-
tive therapies is considered to be related to main-
taining sufcient quantities of a specic factor at the
local site. Thus, studies examining the quantity of
specic growth factors and their associated recep-
tors at a given site during wound healing, such as
a non-critical size defect, may help to establish the
essential factors, both type and quantity, required for
promoting regeneration at healing sites.
Maturation
As with other mineralized tissues, mature cementum
contains several factors that were either secreted by
cementoblasts or absorbed by hydroxyapatite during
root formation (11).
Mineralization
Although several factors have been implicated as
having an important role in controlling mineraliza-
tion, the specic role of individual molecules has yet
to be established. Investigations targeted at mapping
the expression pattern for specic molecules devel-
opmentally, as well as use of knock-out mice, have
enhanced understanding of these molecules in the
mineralization process, including cementogenesis.
Development
As noted in Table 1 several factors that have been
implicated as having a role in regulating mineraliza-
tion may also have other functions. In this context,
the potential roles for bone sialoprotein, osteopontin
and type I and XII collagen in regulating crystal
growth, both during development and in regenera-
tion of periodontal tissues, were discussed under the
heading of adhesion molecules.
In addition to these molecules both osteocalcin
and proteoglycans are considered to be important
for regulating mineralization in various tissues in-
cluding cementum. We and others have demon-
strated the selective expression of osteocalcin to root
lining cells, cementoblasts, with this prole of ex-
pression being maintained in the mature tissue, that
is, periodontal ligament broblasts do not express
osteocalcin. Expression pattern for matrix gla pro-
tein during root development and in mature tissues
has not been studied. The temporal and spatial ex-
pression pattern for osteocalcin during cementogen-
esis (56), and the selectivity of osteocalcin to min-
eralized tissues suggests that it has a prominent role
in root development. Studies by Ducy et al. (61), in-
Molecular and cell biology of cementum
dicate that transgenic mice lacking the osteocalcin
gene develop a phenotype marked by enhanced
mineral density, suggesting that osteocalcin may be
a controller of mineral formation. Thus, it is possible
that osteocalcin is one of the molecules that control
the mineral-to-ligament ratio, allowing for formation
of a periodontal ligament region versus an ankylosis
situation.
Regeneration
As discussed above under adhesion/migration fac-
tors, both osteopontin and bone sialoprotein are ex-
pressed in cells/tissues associated with periodontal
wound healing (125). Also, osteocalcin and types I,
III and XII collagen have been associated with
wound healing and/or stress to the periodontal
tissues associated with orthodontic tooth movement
(238). These molecules, as well as proteoglycans,
most likely play a critical role during wound healing
in balancing formation of cementum and surround-
ing bone, with development of a functional peri-
odontal ligament.
Maturation
Mature cementum contains all of the non-colla-
genous molecules discussed above. Mature ce-
mentum has a high percentage of type I collagen,
with less amounts reported for other collagens, in-
cluding type III and type XII collagen. The small
amount of other collagens identied in cementum
may be produced by periodontal ligament bro-
blasts, where the formed collagen bers are inserted
into cementum.
Factor-mediated cell activities
A key for designing agents for use in regeneration of
tissues is an understanding of the molecular conse-
quences resulting from interactions between factors
and corresponding receptors. Substantial progress
has been made in understanding of regulators of cell
function in health, as well as in diseased states. How-
ever, until recently, few studies have been performed
with cells considered to be associated with root de-
velopment, such as epithelial root sheath cells, fol-
licle cells and root surface cells/cementoblasts due
to lack of availability of such cell types. In contrast,
more information is available on cells associated
with the mature periodontium, such as osteoblasts
and periodontal ligament cells. In this section funda-
mental concepts related to ligandreceptor interac-
tions are presented as a means of stressing the im-
81
portance, as well as the complexities, for under-
standing basic mechanisms regulating cell activities
to designing of regenerative therapies. For example,
using a hypothetical model, it may be possible that
factor A is capable of promoting mineralization of
mature osteoblasts but not of periodontal broblasts
or preosteoblasts. Further, the reason for this may be
related to the lack of appropriate receptors on the
latter cells; however, addition of factor B may prime
these cells so that they now express the receptor re-
quired for responding to factor A.
Provided below is a broad overview of current
knowledge as to growth factorcell interactions, with
a discussion on how this may relate to control of de-
velopment and/or regeneration of periodontal
tissues. For in-depth reviews of this area, the reader
is referred to Sastry & Horwitz (198); Lafrenie & Yam-
ada (123); Force & Bonventre (68); and Ingber (109).
Growth factors have numerous effects on cells de-
pending on both the factor and cell type and stage of
maturation. For example, growth factors can initiate
DNA synthesis, modulate differentiation and alter
the cytoskeleton. The short half-life of growth factors
and their association with extracellular matrix and
growth factorbinding proteins ensure their local ef-
fects. The extracellular matrix molecules and growth
factors exert effects through specic cell surface re-
ceptors, and when the receptor is bound it interacts
with cytoplasmic effector molecules to initiate a
complex cascade of intracellular events leading to an
alteration in gene function (see Fig. 3 and 4 for an
overview of selected pathways).
Receptor tyrosine kinases
Growth factor receptors are prototypic members of
a family of cell surface receptors characterized by an
extracellular binding domain, a single transmem-
brane portion and a large intracellular catalytic do-
main. Specically, those that possess intrinsic tyro-
sine kinase domains belong to the hydrophilic re-
ceptor family and are referred as receptor tyrosine
kinases. The signaling molecules for this group of re-
ceptors include epidermal growth factor, insulin-like
growth factor, broblast growth factor, platelet-de-
rived growth factor, nerve growth factor and insulin.
As the ligand binds to the receptor, the kinase do-
main is autophosphorylated and transmission of the
molecular signal from the ligand to the cytoplasmic
domain of the receptor occurs by a dimerization of
the receptor complex (161). Receptor activation trig-
gers intracellular cascades of protein phosphoryla-
tions that promote protein interactions. These inter-
Saygin et al.
actions result in the transmission of specic signals
to the nucleus capable of controlling gene express-
ion, with ultimate alteration of cell function. A good
example that demonstrates the complexities of these
activities is the mitogen-activated protein kinase
pathway. Mitogen-activated protein kinase phos-
phorylates transcription factors resulting in the acti-
vation of specic genes. Mitogen-activated protein
kinase has 3 isoforms (mitogen-activated protein ki-
nase and extracellular signalregulated kinase-1
and -2), which can be activated in response to
growth factors and other mitogens. Extracellular sig-
nalregulated kinase-1 and -2 proteins are present
in human osteoblasts, human bone marrow stromal
cells, rat osteoblastic cells (ROS 17/2.8 and UMR-
106), and mouse osteoblastic cells (MC3T3-E1) (34).
Insulin-like growth factor-I, broblast growth factor-
2 and platelet-derived growth factor-BB were found
to activate extracellular signalregulated kinase-2.
Receptor tyrosine phosphorylation also promotes in-
teractions with a number of other molecules such as
phospholipase-Cg, non-receptor Src family of ki-
Fig. 3. Putative receptor-cementoblast interactions. derived growth factor; IGF: insulin-like growth factor;
Ligand-receptor binding results in activation of effector FGF: broblast growth factor; PG: prostaglandin; PTH/
molecules that act as intracellular relay systems to trigger PTHrP: parathyroid hormone/parathyroid hormonere-
gene expression. Abbreviations: TGF: transforming growth lated protein. Adapted from Fuller & Shields (71).
factor; BMP: bone morphogenetic protein; PDGF: platelet-
82
nases, tyrosine phosphatases and phosphoinositide-
3-kinase (30, 71).
Several growth factors have been shown to inter-
act with periodontal broblasts and/or osteoblasts
and these include members of the transforming
growth factor-b super family, platelet-derived growth
factor, insulin-like growth factor, epidermal growth
factor, broblast growth factor, prostaglandin E, and
parathyroid hormone/parathyroid hormonerelated
protein. In 1991 Narayanans group isolated a factor
from cementum, which they called cementum de-
rived growth factor (164, 254). In subsequent studies
they reported that cementum-derived growth factor
is an insulin-like growth factor-I-like molecule (107).
Insulin-like growth factor-I has been shown to po-
tentiate the effects of several growth factors and
other molecules on cell activity (81, 197). Similar to
responses noted with growth factors such as insulin-
like growth factor-I, cementum-derived growth fac-
tor was found to cause a transient increase in cyto-
plasmic Ca
2
concentration, promote phosphoinosi-
tol-phosphate hydrolysis, activate the protein ki-
Molecular and cell biology of cementum
nase-C cascade and increase expression of cellular
protooncogenes in gingival broblasts (255).
Growth factor interactions with their specic re-
ceptors, coupled with their complex interactions
with other molecules, including other growth factors
and integrins, result in a complex set of cell re-
sponses (198). Thus, the effect of a growth factor on
a specic cell in vitro may be very different from the
effects of that factor on another cell type in vitro or
in fact on the same cell type in vivo. Few studies
have examined the role of growth factors in regulat-
ing cementogenesis, where more extensive studies
have been performed using animal models, versus
in vitro or in situ models, and these are discussed
later.
Examining literature on in situ models, using
radioautography and 14-day-old rats, Cho et al. (45)
investigated the role of epidermal growth factor on
differentiation of cementoblasts after injecting
125
I-
epidermal growth factor. They reported that during
differentiation of cementoblasts a very low level of
epidermal growth factorbinding sites were present
on the mesenchymal cells in dental follicle proper,
precementoblasts and cementoblasts, indicating the
limited effect of epidermal growth factor on ce-
mentoblast differentiation. However, preosteoblasts,
prechondroblasts, perifollicular cells and mature
periodontal ligament broblasts exhibited epidermal
growth factorbinding sites in vivo (44). Thus, while
interactions between epidermal growth factor and
epidermal growth factor-receptor are not involved
directly in the regulation of cementoblast activities,
these studies, as well as those by other groups (158,
178, 179, 230), indicate that epidermal growth factor
and associated interactions with other cells during
tooth/periodontal development are critical for for-
mation of a functional periodontium.
G-protein-coupled receptors
G-protein-coupled receptors are associated with the
ability of growth factors to activate the mitogen-acti-
vated protein kinase cascade and other tyrosine ki-
nase pathways. G-proteins act as the initial effector
activating substrate for multipass receptors. The
major downstream effector molecules controlled by
G-proteins are adenyl cyclase, phospholipase C,
phospholipase A
2
, phosphoinositide 3-kinase, and b-
adrenegic receptor kinase. The activated second
messengers, such as cyclic adenosine monophos-
phate, diacylglycerol, inositol triphosphate and cal-
cium, are small molecules that amplify receptor-acti-
vated signals. Further, many growth factorG protein
83
Fig. 4: Growth factor signaling cascade via MAPK. Abbrevi-
ations: TK: tyrosine kinase; SOS: son of sevenless; MAPK:
mitogen-activated kinase; MAP2K: mitogen-activated ki-
nase kinase; TF: transcription factor. Note: this is only
one aspect of the GF-signaling pathway.
interactions, such as platelet-derived growth factor
and insulin-like growth factor-II, are known to acti-
vate the mitogen-activated protein-kinase pathway
(148, 197). With regard to cementum, it is known that
cementoblasts express parathyroid hormonerelated
protein receptors (228), and thus it is not surprising
that both parathyroid hormone and parathyroid hor-
monerelated protein promote an increase in cyclic
adenosine monophosphate in these cells (56, 57,
174). Parathyroid hormone/parathyroid hormone
related protein, through G-protein-linked receptors,
evoke multiple parallel signaling events that include
activation of adenyl cyclase (protein kinase-A path-
way), phospholipase C (protein kinase-C pathway)
and cytosolic free calcium transients (225).
Saygin et al.
Serine-threonine receptor kinases
Transforming growth factor-b superfamily molecules
are known to elicit their effects through interactions
with serine/threonine kinase receptors. Subsequent
to their interactions with and activation of the recep-
tor, a group of signaling molecules, Smads, are phos-
phorylated selectively by bone morphogenetic pro-
teins and form heteromeric complexes (195). Some
of these Smads enter the nucleus, where they induce
transcriptional activation and subsequently, alter
cell behavior. Bone morphogenetic proteins have
numerous functions, including regulation of cell
growth, differentiation and apoptosis in a variety of
cell types, such as osteoblasts, neural cells, epithelial
cells (184, 195, 201, 229, 232, 239); however, their ex-
act role in cementum formation is only beginning to
be explored (1). Their potential role in regeneration
of periodontal tissues is discussed later.
Integrins
A required event for development and regeneration
of cementum is attachment of appropriate cells on
the root surface. While several adhesion molecules
have been identied on the root surface at various
stages of root development, such as bronectin, la-
minin, type I collagen, bone sialoprotein, cementum
attachment protein and osteopontin (Table 1, Fig. 1),
the importance of these molecules for controlling
cell adhesion and differentiation during tooth root
formation are not known. Further, certain growth
factors that have been implicated in controlling cell
adhesion, for example, platelet-derived growth fac-
tor-BB and insulin-like growth factor-I, have been
shown to regulate expression of integrins (40) and
proteoglycans (87). Many adhesion molecules inter-
act with cells through specic integrins on the cell
surface. Integrinextracellular matrix binding acti-
vates signal transduction pathways and hence regu-
lates gene expression (71, 130). Particularly central
to integrin signaling are focal adhesion kinases,
which have been reported to bind directly to inte-
grins. A number of other signaling molecules then
bind to focal adhesion kinases and are phosphoryl-
ated by it, linking focal adhesion kinases to the mito-
gen-activated protein kinase pathway and to the 85-
kDa subunit of phosphoinositide 3-kinase (189),
thus linking the integrin signaling pathway with sev-
eral other pathways.
Few studies have focused on the role of these inte-
grins during tooth development (196, 252), and thus
the specic integrins required for cell adhesion at
84
the local root surface site, as well as the ligands in-
volved, remain unknown. Studies by Saito & Naray-
anan (193), demonstrated that molecules extracted
from mature cementum that promote adhesion of
broblasts induce characteristic signaling events,
such as activation of c-fos, focal adhesion kinases
and extracellular signalregulated kinase-2. It is
possible that such molecules may play a role in re-
cruitment of specic cell types to the local site dur-
ing wound healing. Future studies directed at deter-
mining the specic adhesion molecules and sig-
naling pathways regulating cementoblast maturation
will aid in enhancing understanding of root develop-
ment and thus in the design of appropriate therapies
for activating cementoblasts.
Regenerative therapies and
cementogenesis in vivo
This section focuses on preclinical and clinical pro-
gress toward using growth factors for stimulating
periodontal regeneration, with an emphasis on ce-
mentum regeneration. Regulators of periodontal
tissue regeneration that stimulate formation of bone,
periodontal ligament and cementum include many
different agents categorized as follows: I) cell occlus-
ive membranes (including guided tissue regenera-
tion) (242); II) bone replacement grafts (such as
autografts, allografts, xenografts and alloplasts)
(157); III) root conditioning agents (such as citric
acid, ethylenediaminetetraacetic acid) (75); and IV)
growth and attachment factors (such as bone mor-
phogenetic proteins, platelet-derived growth factor
and enamel matrix derivative) (88, 150).
A multitude of studies testing therapies to stimu-
late periodontal repair have been published. How-
ever, the evidence to support the use of these therap-
ies as modulators of complete periodontal regenera-
tion is quite limited. Table 2 highlights studies
focusing on therapeutics measuring cementogenesis
in vivo in both preclinical and clinical settings.
The results of preclinical and clinical studies will
be reviewed, and the ability of these molecules to
not only stimulate cementogenesis but also govern
bone and periodontal ligament regeneration will be
discussed.
Therapies based on platelet-derived growth factor
and insulin-like growth factor
Some of the earliest in vivo studies assessing the role
of growth factors on periodontal regeneration fo-
cused on a combination of platelet-derived growth
Molecular and cell biology of cementum
Table 2. Agents demonstrated to promote cementogenesis in preclinical and/or clinical studies
Evidence of cementogenesis
Therapy Preclinical (animal) Human histology References
Growth factors
Platelet-derived growth factor or platelet-derived Yes No (79, 100, 134, 190)
growth factor/insulin-like growth factor-I
Bone morphogenetic protein 2 Yes No (118, 207)
Bone morphogenetic protein 3 No Yes (23)
Bone morphogenetic protein 4 Yes No (186)
Bone morphogenetic protein 7 (OP-1) Yes No (80)
Bone allografts Yes Yes (24, 25, 26, 183)
Xenogenic bone grafts Yes Yes (32)
Autogenous bone grafts Yes Yes (70, 97)
Citric acid demineralization Yes Yes (3)
Enamel matrix derivative Yes Yes (88, 94)
factor alone or combined with insulin-like growth
factor-I. Results using natural disease lesions in dogs
and ligature-induced lesions in nonhuman primates
showed that this growth factor combination pro-
moted formation of new bone, cementum and peri-
odontal ligament (79, 134, 190).
Park et al. reported results in a dog model using
platelet-derived growth factormodulated guided
tissue regeneration therapy. Findings from this study
demonstrated the promotion of new bone, ce-
mentum and periodontal ligament, measured at 5
and 11 weeks after platelet-derived growth factor/
guided tissue regeneration treatment in class III fur-
cation defects (177). Interestingly, when platelet-de-
rived growth factor was applied to tooth roots di-
rectly (following citric acid demineralization of the
root surface without the use of a barrier membrane),
extensive ankylosis ensued (46). The ankylotic union
of bone to tooth without intervening cementum and
periodontal ligament was found in 100% of the
specimens evaluated at 5 weeks and 83% of the
specimens at 8 weeks. Further investigations are
needed to explore this result, since platelet-derived
growth factor-induced ankylosis has not been de-
scribed in other studies.
The rst human clinical trial testing the safety
and efcacy of recombinant human platelet-de-
rived growth factor/recombinant human insulin-
like growth factor-I was completed in 1997 (100).
This study examined 38 patients possessing moder-
ate to severe periodontal disease treated with a)
150 mg/ml each of recombinant human platelet-de-
rived growth factor-BB and recombinant human in-
sulin-like growth factor-I in a methylcellulose ve-
hicle or b) vehicle alone or c) surgery alone. The
results revealed patients treated with recombinant
85
human platelet-derived growth factor/recombinant
human insulin-like growth factor-I responded with
42.5% osseous defect ll, while the control group
consisting of pooled vehicle and surgery alone
demonstrated only 18.5% osseous defect ll. The
growth factors were shown to be safe and well tol-
erated by the subjects. Furcation lesions responded
most favorably to treatment with nearly a four-fold
increase in bone volume compared with paired
controls. These results in humans were found to
be highly consistent when compared to preclinical
studies in nonhuman primates (77). Ongoing
studies, using these growth factors with a variety
of delivery systems, should provide the information
required to determine the clinical feasibility of
using platelet-derived growth factor/insulin-like
growth factor-I for regeneration of periodontal
tissues in humans.
Transforming growth factor-b family members:
bone morphogenetic proteins 2, 3 (osteogenin),
4, and 7 (OP-1)
The bone morphogenetic proteins have been evalu-
ated extensively in orthopedic models for their abil-
ity to induce osteogenesis (181). Bone morphogen-
etic protein-2 is the most thoroughly researched
member of the transforming growth factor-b super-
family for the promotion of periodontal and peri-
implant bone regeneration (48, 91, 92, 99, 118, 191,
206208). This molecule has demonstrated potent
effects in stimulating cementogenesis. Sigurdsson
et al. reported the effects of recombinant human
bone morphogenetic protein-2 on periodontal re-
generation and found that bone morphogenetic
protein-2 applied in synthetic bioabsorbable poly-
Saygin et al.
mer greatly stimulated new bone and cementum
formation (207). These results were achieved 8
weeks following bone morphogenetic protein-2 ap-
plication. Close to 95% of the bone in surgically
created class III furcation lesions was regenerated.
However, a near 4-fold increase in ankylosis was
found in bone morphogenetic protein-2-treated
sites as compared to vehicle. Ripamonti et al. dem-
onstrated potent stimulation of cementum and
bone regeneration in class II furcation defects in
baboons using topical bone morphogenetic pro-
tein-4 application (186).
The rst human study using a bone morphogen-
etic protein to promote periodontal regeneration
utilized a single application of bone morphogenetic
protein-3 (osteogenin) combined with demineral-
ized bone allograft in a submerged tooth model
(23). The investigators found increased bone and
cementum deposition around periodontally in-
volved submerged teeth with bone morphogenetic
protein-3 treatment as assessed by human his-
tology. However, the bone morphogenetic protein-
3 was not signicantly better than carrier alone
(bone graft group). Additionally, pinpoint ankylosis
was observed in submerged teeth grafted with bone
morphogenetic protein-3 plus bone grafts. More re-
cently, our group demonstrated closure of class III
furcation defects with the application of bone mor-
phogenetic protein-7/OP-1 applied to periodontal
defects, using an animal model (80). Heightened
stimulation of the formation of cellular regenera-
tive cementum could be noted at 8 weeks post
bone morphogenetic protein-7/OP-1 application.
Interestingly, some root surfaces exhibited dentin
resorptive pits with regenerative cementum form-
ing over the irregular root surface (Fig. 5). Another
interesting observation was that ankylosis was not
observed in areas where regenerative cementum
occurred. While this needs to be examined more
carefully, this suggests that identication of factors
that can promote new mineralization consistently
on the root surface is an important area for re-
search development.
Transforming growth factor-b
The rst published report examining transforming
growth factor-b in periodontal defects examined its
role in combination with insulin-like growth factor-
II and basic broblast growth factor (200). The re-
sults in surgically created fenestration defects in
dogs failed to show a benet by the application
of these factors. The authors reported the use of
86
nanogram quantities of growth factor, which may
in part explain the negative results. More recently,
Wikesj et al. reported the use of transforming
growth factor-b in periodontal repair and stimula-
tion of cementum formation (244). They tested the
ability of transforming growth factor-b1 coupled
with expanded polytetrauoroethylene barriers to
stimulate periodontal regeneration. The results of
the study failed to reveal any benet with the com-
bined therapy. However, the authors did not use a
group containing transforming growth factor-b
without barriers, and the exclusion of periosteal
cells may have been a factor in results achieved.
Cochran et al. demonstrated an inhibition of bone
promotion around implant xtures when bone
morphogenetic protein-2 was used in combination
with barrier membranes (48). Mixed reports on the
ability of transforming growth factor-b to promote
regeneration of mineralized tissues in orthopedic
models have been reported as well (33). At the
present time, transforming growth factor-b needs to
be further explored for its potential use in recon-
structive periodontal therapy.
Enamel matrix derivative
Enamel matrix derivative, where the principal pro-
tein is amelogenin, is approved for human use inter-
nationally. Enamel matrix derivative has been evalu-
ated, in a variety of preclinical and clinical settings,
for its ability to stimulate cementum and peri-
odontal attachment structures. In a preclinical buc-
cal dehiscence model in nonhuman primates, Ham-
marstrm et al. tested the ability of enamel matrix
derivative to affect periodontal wound healing (90).
When compared with carrier and ethylenedi-
aminetetraacetic acid root conditioning, enamel ma-
trix derivative treated defects demonstrated signi-
cant enhancements of bone, cementum and peri-
odontal ligament. In human studies, enamel matrix
derivative has demonstrated partial regeneration by
human histology (94), safety in a multicenter trial of
10 test centers and 107 patients, and efcacy as
shown by a placebo-controlled human trial using 33
subjects with paired intrabony defects (95). The later
study assessed enamel matrix derivative therapy
coupled with modied Widman ap surgery on peri-
odontal wound healing as measured by clinical
attachment level change and subtraction radi-
ography (95). The enamel matrix derivative therapy
promoted 66% defect ll 36 months post-therapy,
while paired control defects failed to show a change
in radiographic bone level. Thus, this study con-
Molecular and cell biology of cementum
Fig. 5. BMP7/OP-1 stimulates cementogenesis in vivo: larly from the root surface are anchored to the adjacent
A. A representative lesion treated with 7.5 mg/g recombin- alveolus (Goldners, 200 magnication). C. Occasional
ant human OP-1 in a collagen carrier. Pronounced new resorptive pits found on the dentinal surface contiguous
bone formation can be seen with numerous embedded with the adjacent cemental dentinal interface (Goldners,
osteocytes in the mineralized matrix (Goldners, 40 mag- 400 magnication). The interface in C can be seen under
nication). B. Cellular regenerative cementum at high ultraviolet light to further demonstrate the cemental/den-
magnication of the lower box in A. A thick layer of ce- tinal line of reversal (ultraviolet unstained section, 400
mentum with collagen-like bers oriented perpendicu- magnication).
cluded that topical enamel matrix derivative appli-
cation in conjunction with modied Widman ap
stimulates periodontal regeneration and is stable for
up to 36 months. Expanded studies in larger patient
populations will be needed to further assess the ef-
fects of enamel matrix derivative treatment in intra-
bony defects.
Delivery systems
The rate limiting step in re-engineering of peri-
odontal structures (including cementum) is the tar-
geting of agents/cells to the tooth root surface. The
healing of a periodontal wound is complicated by
several factors that limit predictable delivery of
agents to the root surface, such as: 1) the transmu-
cosal environment of the mineralized tooth surface
traversing keratinized gingiva; 2) a complex micro-
biota contaminates wounds at the soft-hard tissue
interface and may affect the release kinetics of deliv-
ery devices; 3) occlusal forces on the tooth complex
in transverse and axial planes may modulate the
healing response and disrupt the stability of delivery
devices; and 4) the complexity of the attachment ap-
paratus includes several stromal/cellular interac-
tions that will require proper orientation of vehicles
within these wounds. Hence, the use of various poly-
mer delivery systems will need to circumvent these
challenges. To date, various biodegradable polymeric
matrices have been developed for use in guided
tissue regeneration (242). The above characteristics
of periodontal wounds place many limitations on
the successful use of these devices. For the delivery
87
of bioactive molecules including growth factors,
genes or cells, materials such as copolymers of poly-
glycolic acid and polylactic acid have been utilized
as delivery vehicles in medicine and dentistry. A
major focus of research in tissue transplantation,
growth factor and gene transfer is the development
of novel delivery systems to allow the extended re-
lease of these agents to the target tissue (tooth root
surface).
Models to study cementogenesis
Investigations targeted at understanding the cellular
and molecular mechanisms controlling develop-
ment and regeneration of periodontal tissues have
utilized both in vitro and in vivo models. Commonly
used animals for regenerative studies include ro-
dents, canines, felines and nonhuman primates (78,
122, 185, 190). Investigations focused on determin-
ing regulators of tooth/periodontal development
often use rodents and follow molar and/or incisor
development (5, 29, 56, 96, 135, 140, 143, 187, 192,
232, 236, 237, 240, 253). In vitro models include cell
cultures where cells are obtained from animal tissues
and cells can be manipulated in various ways to
mimic the in vivo environment, such as growing
cells on or within selective matrices or using a var-
iety of co-culture models (49, 69, 117, 132, 168).
While in vivo models reect the complexities of
host-cell interactions, and thus may more accurately
reect the regenerative activities in humans (80),
versus in vitro models, there are signicant limi-
tations. Thus, there is a need for both in vitro and in
Saygin et al.
vivo studies, where in vitro models, using cell sys-
tems, provide the tools required to understand the
response of cells to specic factors and the molecu-
lar factors controlling these responses, and in vivo
models provide the tools required to establish proof
of concept. Limitations of in vivo models include
the inability to design a model that harbors the exact
type of defect seen in human disease. Further, many
models use an acute defect which, while valuable for
determining whether a factor does elicit a response,
may not reect the response for chronic situations,
such as those found with most individuals who have
periodontal disease. Moreover there are distinct gen-
etic, anatomic, biochemical, immune and microbial
differences between the species. In attempts to over-
come these drawbacks genetically engineered ani-
mals, as well as cells in vitro and in vivo are being
used. Discussed below are animal and cell models
that are being used to understand periodontal dis-
ease or that have potential for use in future studies.
In vitro models
Excellent studies at the light and electron micro-
scopic level have provided a detailed analysis of ce-
mentum at various stages of development and also,
during regeneration of periodontal tissues sub-
sequent to disease (20, 21). Further, immunocyto-
chemical studies and in situ hybridization studies
have provided information as to the factors ex-
pressed by cells associated with the periodontium.
However, these studies present only indirect infor-
mation as to the factors critical to formation of the
periodontium (19, 22, 29, 56, 137, 138, 140, 142, 144,
153, 216, 217, 227). As discussed later, transgenic/
knock-out animals offer another tool to assist in de-
termining the role of specic molecules in control-
ling tissue function, but with limitations since such
animals often do not survive, or show no phenotype,
or show a complex phenotype that requires further
analysis. Cell cultures provide an additional tool
where advances in cell and molecular techniques
allow for selected manipulation of specic cell types.
In addition, cells isolated in culture can be reintro-
duced into a specic site in animals and the activity
of the cell type conrmed in vivo.
An important aspect of cell culture is to be able to
describe the cell phenotype at some level prior to
isolation, since with passage cells in culture often
lose their phenotype. In this regard, studies focused
on establishing factors expressed by cementoblasts
and precementoblasts (follicle cells) in situ allowed
for the isolation of cementoblasts where cells in vitro
88
could be analyzed to ensure that they expressed the
same genes identied for these cells prior to iso-
lation (see Fig. 1, selective stage markers). Fortu-
nately, using these tools our laboratory has success-
fully isolated and cultured cells from the developing
root surface of mice using a variety of techniques
(5457, 212). Briey, as a rst approach, a mixed
population of periodontal ligament cells and ce-
mentoblasts were isolated by collagenase-trypsin di-
gestion, and cells were shown to exhibit properties
associated with these cells in vivo, however cells did
not survive continued passages (56). Therefore, sev-
eral approaches were used to establish cells that
would survive passage and maintain phenotype, in-
cluding immortalization of primary cell cultures
using wild-type SV40 (28), using immortalized trans-
genic mice, immorto-mice (54, 57, 111, 112, 212),
and using osteocalcin promoterdriven SV40 Tag
mice (35). The advantage of the latter is that only
root surface cells expressing osteocalcin, cemento-
blasts, will survive in vitro, thus excluding peri-
odontal ligament cells from the population. The het-
erogeneous cell populations also are of value since
they reect the local environment in vivo and by
subcloning mixed populations, both periodontal
ligament cell lines and cementoblast populations
can be established. Cementoblasts in vitro express
transcripts for bone sialoprotein, osteocalcin, osteo-
pontin, alkaline phosphatase, osteoblast-specic
transcription factor, parathyorid hormone/para-
thyroid hormonerelated protein receptor 1 and type
I collagen (54, 56, 57). In addition, these cells re-
spond to parathyroid hormone and vitamin D in a
fashion similar to that noted for osteoblasts and also
promote mineral nodule formation both in vitro and
in vivo (54). Additionally, these cells were found to
be responsive to several growth factors, including
platelet-derived growth factor and insulin-like
growth factor (unpublished data).
In vitro models with human cementoblasts, as re-
ported by Grzesik et al. (86), will provide additional
tools for understanding the behavior of periodontal
tissue. While several groups have been successful in
isolating and characterizing cultured human peri-
odontal ligament cells, it is known that these are
mixed populations, and also that they change pheno-
type with culture. Thus, using a similar approach of
rst establishing cell phenotype in vivo and then isol-
ating, immortalizing and subcloning human popula-
tions will allow for studies on these cells. Unfortu-
nately, markers selective for periodontal ligament
cells have not been established. Nevertheless, immor-
talized periodontal ligament cell lines are available,
Molecular and cell biology of cementum
where future studies will help to clarify if these cells
reect the in vivo cell type (98).
In addition, cells from a human cementoblastoma
were isolated and cultured. However, since the ce-
mentoblastoma is poorly dened, the exact cells
obtained for culture are unclear. Cells cultured from
this tumor produced bone sialoprotein, cementum
attachment protein and collagen types I and V and
also promote mineralized nodules in vitro (6, 7). As
specic markers for cementoblasts are established,
these cells may prove to be an excellent source for
human cells. Importantly, with the availability of cell
cultures, critical issues regarding the mechanisms
and factors regulating cementoblast function, in-
cluding identication of genes expressed selective by
cementoblasts, can be addressed. Beyond determin-
ing the properties of cementoblasts in vitro, these
cells can be used for targeted gene therapy as dis-
cussed later (166).
In vivo: genetically engineered animals
Reverse genetic techniques, including gene knock-
outs and transgenesis, allow dened mutations to be
introduced into the mouse genome and provide in-
sight into gene function (84, 106, 205). The mouse is
particularly useful as its genome is very well char-
acterized, and genes can therefore be manipulated
withrelative ease (72). Understanding the factors con-
trolling periodontal diseases and specically ce-
mentogenesis may benet from the use of transgenic
or knock-out animals, as well as animals with spon-
taneous mutations that reect systemic diseases or
syndromes associated with alterations in periodontal
tissues, such as Pagets disease, hypophosphatasia,
Table 3. Methods for delivering genes into mammalian cells and likely applications in gene therapy
Application in gene therapy
Transient or
Method Ex vivo In vivo stable expression
Viral
Retrovirus ? Stable
Adenovirus Transient
Adeno-associated virus ? Stable
Herpes virus Not predictable
Vaccina virus Transient
Polio virus Transient
Sindbis or other RNA viruses Transient
Nonviral
Ligand-DNA conjugates Transient
Adenovirus-ligand-DNA conjugates Transient
Lipofection Transient
Direct injection of DNA Transient
CaPO
4
precipitation Stable
(): Major application, (): some application. (): little or no application. (?): safety concern. Adapted from Mulligan (162).
89
cleidocranial dysplasia, hypopituitarism and osteo-
petrosis. Animal models demonstrating alterations in
tooth structure most often are reported as failure of
tooth eruption, where the major defect is related to
lack of osteoclast activity, and include op/op mice (2,
64, 163, 222), c-fos knock-outs (110, 114, 243), src
knock-outs (27, 220), and osteoprotegerin ligand
knock-outs (120, 223). Rescue attempts toward re-
placement of missing factors have provided a means
of maintaining animals that would normally not sur-
vive. For example, parathyroid hormone/parathyroid
hormonerelated protein knock-out mice have severe
alterations in skeletal development and do not sur-
vive (66, 115, 124). Rescue of these animals by a trans-
gene for chondrocytic parathyroid hormonerelated
protein expression failed to recover eruption and root
formation, while rescue using a keratin-driven trans-
gene/parathyroid hormonerelated proteincorrected
the defect of eruption and tooth formation (180).
Another area of focus has been to overexpress or
underexpress selective genes at various stages of de-
velopment and then to determine the effect of these
manipulations on animal function. Ibaraki-OConn-
or et al. (105) have established a transgenic mouse
line to investigate the function of amelogenin during
mineralization. Importantly, future studies using this
cell line may provide insight as to the role of amelog-
enin-like molecules, including enamel matrix deriva-
tive, in controlling cells associated with the peri-
odontium.
Cell applications: regional gene therapy
The application of putative molecules to induce or
modulate periodontal regeneration is an area of in-
Saygin et al.
tensive interest. However the short half-lives of these
molecules at the healing site may reduce their effects
in vivo. Therefore, methods that provide stability of
exogenous molecules at the healing site may be ad-
vantageous toward maximizing wound repair. The
ability to transfer genetic material into specic cells
offers an approach that may help sustain the effect
of the targeted factor. Several investigators have fo-
cused on developing ideal methods for delivery of
genes to cells for subsequent use in in vivo models.
Gene transfer can be performed with strategies for
either ex vivo or in vivo transfer of the desired trans-
gene. Inex vivo transfer, cDNAis transferred to cells in
culture, and the genetically modied cells are ex-
panded and then administered to the recipient site
(162). For example, bone marrow cells can be re-
moved from an individual, transduced ex vivo and
then the genetically modied cells reimplanted to the
site of interest (127). The alternative is the in vivo
technique, where the gene is transferred directly into
the target tissues of the recipient by microseeding of
plasmid or viral DNA (77). The attractiveness of using
regional gene therapy to induce repair and formation
is that genes can be delivered to the appropriate ana-
tomic site, and the duration of protein expression can
be determined by selecting the appropriate vector
and/or promoter (162). Both viral and nonviral
methods are used for gene transfer (Table 3). In the
case of retroviral and adeno-associated viral vectors,
the transferred DNA sequences are stably integrated
into the chromosomal DNA of the target cell. In gen-
eral, this approach is used for ex vivo application.
However, incertainsituations, gene expressionis high
but transient, and such conditions favor in vivo trans-
fer. Althoughretroviral gene transfer is ideal for ex vivo
applications, several features of the gene transfer
method may limit its applicability, particularly with
regard to in vivo applications. The entry of the retro-
virus depends on the existence of the viral receptor. In
addition, the replication of the target cell is necessary
for proviral integration to occur.
The most important advance for viruses as gene
transfer vectors was the generation of packaging
cells that permit the productionof hightiters of repli-
cation-defective recombinant virus, free of wild-type
virus; also called gutless viruses (63, 93, 131, 160).
Adenoviruses are capable of efciently infecting non-
dividing and dividing cells and expressing large
amounts of gene products (162). The transient nature
of adenoviruses allows them to be used in high titers
since their effect will gradually diminish and remain
unintegrated (as extrachromosomal DNA) due in part
to a T-cell-mediated immune response.
90
The gene transfer approach presents an attractive
alternative to the conventional topical application of
molecules to the complex periodontal wound site.
The use of viral constructs containing one or more
response modier transgenes for long-term delivery
may modulate the cellular response in the periodon-
tium. For these studies, the rst step is to determine
the gene transfer efciency of putative cells isolated
from the periodontium, such as periodontal liga-
ment cells or cementoblasts. Although the mechan-
isms that coordinate major regenerative events re-
main obscure, migration, proliferation, attachment,
differentiation and maturation of participating cells
can be modulated by gene transfer methods. Factors
that modulate angiogenesis, proliferation, attach-
ment and differentiation of the cells may be deliver-
ed into the wound by gene therapy. The use of gene
transfer techniques should enable one to modulate
periodontal regeneration, as well as assist in en-
hancing understanding of the mechanisms involved
in wound healing.
Future directions: unknowns
This is a dynamic time for researchers and clinicians
devoted to optimizing periodontal/implant regene-
rative therapies. The explosion in understanding of
regulators of cell function, coupled with tools that
allow researchers to engineer cells so as to express
specic factors, added to improved delivery systems
for controlling release of cells/factors at a given site,
now allows treatment modalities to be designed
based on sound scientic data. Clearly, a rst step is
evaluating engineered cell types/delivery systems in
in vitro and in vivo models. This would include
examining their ability to enhance cell proliferation
and/or mineral nodule formation and/or specic os-
teoblast- and cementoblast-associated genes in vitro
and formation of new bone, new root surface min-
eral and a functional periodontal ligament region in
vivo. Information gained from these studies should
provide the foundation required for designing more
predictable regenerative therapies when compared
with those available at present.
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