regeneration of periodontal tissues: cementogenesis · pdf fileregeneration of periodontal...
TRANSCRIPT
Regeneration of periodontaltissues: cementogenesis revisited
MARGARITA ZEICHNER-DAVID
Virtually all types of periodontal disease are caused
by periodontal pocket infections, although several
other factors, including trauma, aging, systemic dis-
eases, genetics, etc., can contribute to the destruction
of the periodontium (1, 18, 31, 52, 60, 107, 128, 127,
194). Repair of the periodontium and the regener-
ation of periodontal tissues remains a major goal in
the treatment of periodontal disease and is an area
still in need of major research attention, as recently
stated by the American Academy of Periodontology
(260). In general, to achieve complete tissue regen-
eration and repair, it is necessary to recapitulate the
process of embryogenesis and morphogenesis in-
volved in the original formation of the tissue. In the
case of the periodontium, complete periodontal re-
pair entails de novo cementogenesis, osteogenesis
and the formation of periodontal ligament fibers.
Current strategies for periodontal repair are based on
anti-infectious measures such as scaling and root
planing, guided tissue regeneration (with or without
bone grafts) or the use of growth factors, none of
which fully restore the architecture of the original
periodontium. Several different approaches involving
tissue engineering are currently being explored to
achieve complete, reliable and reproducible regen-
eration of the periodontium. As tissue engineering is
defined as the science that develops techniques
(based on principles of cell and developmental bio-
logy) for fabricating new tissues to replace or regen-
erate lost tissues (205), it is important to understand
the formation of specific tissues, the physico-chem-
ical characteristics of the tissues and the molecular
events leading to the normal function of the tissues.
Development of the periodontium
The periodontium can be defined as �an intricate
mosaic of cells and proteins that is primarily
responsible for the attachment of teeth in the oral
cavity� (144). Several excellent reviews have been
published describing the embryonic lineage of the
principal periodontal tissues (cementum, periodontal
ligament, gingiva and alveolar bone), as well as the
cells and extracellular matrix components of the
periodontium (10, 13, 14, 21, 19, 46, 45, 51, 71, 80, 82,
144, 158, 185, 186, 193, 212, 214, 243, 244, 245).
Formation of the periodontium is initiated with
the process of root formation where, following
crown formation, the apical mesenchyme continues
to proliferate to form the developing periodontium,
while the inner and outer enamel epithelia fuse
below the level of the cervical enamel to produce a
bilayered epithelial sheath, termed the Hertwig’s
epithelial root sheath. As these cells divide, there is
an apical migration of the Hertwig’s epithelial root
sheath cells through the underlying dental ectome-
senchymal tissues, dividing them into the dental
papilla and the dental follicle (Fig. 1). As the root
develops, the first radicular mantle dentin is formed
and the epithelial sheath is fenestrated. It is believed
that cells of the Hertwig’s epithelial root sheath
migrate away from the root into the region of the
future periodontal ligament where they re-associate
to form the Epithelial Rest of Malassez. However,
not all Hertwig’s epithelial root sheath cells migrate
into the periodontal ligament site; a few undergo
apoptosis and some remain in the root surface
(108).
Although it is accepted that the Hertwig’s epithelial
root sheath plays an important role in root develop-
ment, the precise nature of its role remains contro-
versial. In 1940, Schour & Massler suggested that the
major function of the Hertwig’s epithelial root sheath
was to induce and regulate root formation, including
the size, shape and number of roots (244). Other
investigators suggested that the role of the Hertwig’s
epithelial root sheath was to induce the differentiation
196
Periodontology 2000, Vol. 41, 2006, 196–217
Printed in Singapore. All rights reserved
Copyright � Blackwell Munksgaard 2006
PERIODONTOLOGY 2000
of odontoblasts to form the root dentin (183, 182, 222,
243, 251), or to differentiate dental sac cells into
cementoblasts (181). The current notion states that
Hertwig’s epithelial root sheath cells produce the
basement membrane containing chemotactic pro-
teins, which serve to direct the migration of prece-
mentoblast cells (140, 141, 182, 235, 251) and to
induce cementoblast differentiation (191, 232, 234).
Amongst the basement membrane molecules are
several extracellular matrix proteins, growth factors,
enamel proteins and adhesion molecules, such as a
collagenous-like protein, known as cementum
attachment protein (CAP), which has chemotactic
potential capable of recruiting putative cementoblast
precursors (11, 149, 156, 196, 275). In the second
stage of cementogenesis (when the tooth reaches
occlusion and cellular cementum is formed), the
proliferation of cells of the Hertwig’s epithelial root
sheath is considerably reduced, and some cells are
entrapped in the newly formed mineral where they
may influence phenotypic changes in the dental sac
cells (252). It is also suggested that Hertwig’s epi-
thelial root sheath cells undergo epithelial–mesen-
chymal transformation to become functional ce-
mentoblasts in charge of producing the acellular
cementum (251, 275).
The gingival tissues appear to be derived from both
the oral mucosa and the developing tooth germ (135).
It has been suggested that the dental follicle (con-
nective tissue surrounding the developing teeth)
gives rise to the fibroblasts forming the periodontal
ligament as well as to the alveolar bone and
cementoblasts (45, 136, 186, 243), all of which have
a common neural crest origin (34). Therefore, it is
postulated that there are different types of
cementoblasts: those originating from the Hertwig’s
epithelial root sheath via epithelial–mesenchymal
transformation and which form the acellular
cementum; and those derived from the dental follicle,
which form the cellular cementum (9, 19, 105, 251,
275). It is also believed that progenitors for perio-
dontal ligament, osteoblast and cementoblast cells
adopt a paravascular location in the periodontal
ligament, and these cells, which exhibit some fea-
tures of stem cells, can regenerate functional tissues
when the need arises (150–153, 195). Periodontal
Fig. 1. Root development and periodontium formation.
Histological sections of 7-day postnatal mouse mandibu-
lar molars showing the initial development of the root by
formation of the Hertwig’s epithelial root sheath. At the
14-day postnatal time-point, apical migration of the roots
continues, and there is formation of the periodontium
with cementum, periodontal ligament and bone. Am,
ameloblasts; C, cementum; D, dentin; Ds, dental sac;
HERS, Hertwig’s epithelial root sheath; Od, odontoblasts;
PDL, periodontal ligament.
197
Regeneration of periodontal tissues: cementogenesis revisited
ligament stem cells have recently been isolated from
the human periodontium (162, 224, 225).
The Epithelial Rest of Malassez cells remain in the
periodontal ligament throughout life, suggesting that
they have important, although yet unknown, func-
tions, rather than just being leftover structures. Roles
attributed to the Epithelial Rest of Malassez cells
range from bad to good. The Epithelial Rest of
Malassez cells are held responsible for the formation
of periodontal cysts and tumors as a result of peri-
apical inflammation associated with pulpal necrosis
(26, 57, 77, 176, 226, 242). It has also been suggested
that Epithelial Rest of Malassez cells contribute to the
formation of the periodontal pocket because of their
continuum with the junctional epithelium (176, 238).
Some studies report the ability of Epithelial Rest of
Malassez cells to resorb bone and extracellular mat-
rix, and thus implicate the cells in root resorption (15,
75, 122). On the other hand, it has also been sug-
gested that the cells of the Epithelial Rest of Malassez
may protect the root from resorption (259). The
finding of Epithelial Rest of Malassez cells being
closely associated with neural endings suggests that
they have a role in the development of periodontal
ligament innervation (126). Studies performed with
1-hydroxyethylidene-1,1-bisphosphonate, a drug that
interferes with homeostasis in the periodontal liga-
ment, showed a severe reduction in the width of the
periodontal ligament with the development of anky-
losis, which was repaired after discontinuing the
administration of 1-hydroxyethylidene-1,1-bisphos-
phonate (261). As the study did not detect a change in
the number of Epithelial Rest of Malassez cells post-
treatment, it was suggested that cells of the Epithelial
Rest of Malassez are unlikely to play an important
part in the homeostasis of, and may not be a prere-
quisite for, the repair and maintenance of the perio-
dontal ligament. On the other hand, the Epithelial
Rest of Malassez cells secrete hyaluronic acid, which
contributes to the formation of the loose connective
tissue characteristics of the periodontal ligament
(155). Cells of the Epithelial Rest of Malassez react to
mechanical stress, like that associated with ortho-
dontic tooth movement, by increasing their prolifer-
ation rate and cell size (27), and thereby help to
maintain the space between the periodontal bone
and cementum to avoid ankylosis (134). The in-
creased activity of the Epithelial Rest of Malassez
cells is consistent with their putative role on collagen
turnover in the periodontal ligament, which is
accelerated during tooth movement (241), and during
cementum repair in areas of root resorption (24). It is
suggested that the Epithelial Rest of Malassez cells
may negatively regulate root resorption and induce
acellular cementum formation (56). In addition, cells
of the Epithelial Rest of Malassez may help in ce-
mentum repair because of their ability to activate
matrix proteins, such as amelogenin, which are also
expressed during tooth development (76, 81).
In summary, based on the information presented, it
appears that the developed or �adult� periodontiumretains its potential for repair/regeneration in the form
of cells of the Epithelial Rest of Malassez, progenitor
cells and stem cells, which can be induced to differ-
entiate into cementoblast, osteoblast or periodontal
ligament cells to regenerate periodontal tissues.
Molecular factors involved inperiodontal development
It is well known that tooth development is regulated by
temporal- and spatial-restricted reciprocal epithelial–
mesenchymal interactions. A number of genes that
play a crucial role in tooth development have been
identified and include growth factors and their
receptors, such as transforming growth factor b-1and )2, bone morphogenetic protein-2 and )4(BMP-2, )4), activins, fibroblast growth factor-4, )8and )9 (FGF-4, )8, )9), hepatocyte growth factor, and
midkine and transcription factors, such as the home-
obox genes (Msx1, Msx2, Dlx1, Dlx2, Dlx3, Otlx2,
Barx1), Pax genes (Pax9 and Pax6), and Lef1, Gli2/Gli3
and Shh (40, 100, 192, 249, 274). It has been docu-
mented that growth factors are involved in establish-
ing the presence, number, site, size or shape of teeth.
The availability of knockout mice has provided critical
information on some growth factors that are deter-
minants of early tooth development. However, little
information is currently available on the growth and
transcription factors involved in the later stages of
tooth development, such as root development. Al-
though one can assume that the same epithelial–
mesenchymal interactions will take place between the
Hertwig’s epithelial root sheath and the underlying
�root�mesenchyme, and all or someof the samegrowth
factors will be involved in root formation, these issues
have been only minimally addressed. Transforming
growth factorb-1 and its receptors (58, 59), andBMP-2,
)3 and )7 (249), have been identified in cemento-
blasts, periodontal ligament and alveolar bone, and
BMP-2, )4 and MSX-2 have been reported in the
Hertwig’s epithelial root sheath (266). Fibroblast
growth factor-2 (143), receptors for epidermal growth
factor (42) and growth hormone (270) have been
detected in periodontal tissues. However, the pub-
lished studies are all descriptive and do not provide
198
Zeichner-David
information as to the function of these growth factors
in periodontium development. Furthermore, the
transforming growth factor-b1-knockout mouse dis-
plays no apparent defects in tooth and root develop-
ment (39), thus excluding a role for this factor in these
processes.On theotherhand, byusing transgenicmice
that express the BMP inhibitor, noggin, driven by the
keratin 14 promoter (K14-noggin), we recently dem-
onstrated that BMPs are important for proper root
morphogenesis. When the function of BMPs is re-
pressed, the transgenic mice demonstrate a delay in
tooth development, lack of enamel formation and
abnormally shaped roots (198). Insulin-like growth
factor-I receptor has been demonstrated in the Her-
twig’s epithelial root sheath, and in vitro experiments
suggest that insulin-like growth factor-I receptor plays
a role in the proliferation and elongation of the Her-
twig’s epithelial root sheath, which is critical for root
development (55).
Transcription factors associated with root develop-
ment include twomembers of the homeobox family of
transcription factors: Dlx2 andDlx3. The expression of
Dlx2by theHertwig’s epithelial root sheathduring root
development was demonstrated using Dlx2/LacZ
transgenic mice (132). Although these studies are only
suggestive of a role of Dlx2 in root development, it was
of interest that theDlx2 knockoutmice showednormal
teeth, while the Dlx1/Dlx2 knockout mice lacked
maxillarymolars (253). The involvementofDlx3 in root
development comes from the phenotype expressed by
patients affected with the genetic disease, tricho-
dento-osseous syndrome, which presents root defects
as well as defects in hair, bone and enamel. A deletion
of 4 bp in the Dlx3 gene, which causes a frameshift
mutation and premature codon termination, resulting
in an altered protein, were identified in a family with
tricho-dento-osseous syndrome (199). We recently
reported the importance of the Nfi-c transcription
factor in root development. Nfi-c knockout mice ap-
pear normal, except that they exfoliate their teeth
shortly after eruption. These mice show a lack of roots
of both mandibular andmaxillary teeth, and therefore
their teeth have no bone attachment. Histological
analysis indicated a normal crown, enamel and dentin
formation, and although there is initial formation of
the Hertwig’s epithelial root sheath and a budding
root, no further development occurs of the roots, ce-
mentum and periodontal attachment apparatus (239).
Cementum composition
In order to understand the process of cementogenesis,
it is important to determine the composition of
cementum. As in bone and dentin, the major com-
ponent of cementum is collagen (16). The expression
of noncollagenous proteins that stimulate cell migra-
tion, attachment, proliferation, protein synthesis and
mineralization during root formation has been
reported by several investigators (38, 142, 147). In the
early stages of root development, immunohisto-
chemical techniques have shown the expression of
multifunctional proteins, such as laminin and
fibronectin (140). These proteins, as well as other
proteins extracted from cementum (173), are initially
believed to function as chemo-attractants. Laminin
and fibronectin can also function as adhesion pro-
teins, together with tenascin (137), bone sialoprotein
(38, 142), osteopontin (25), and a 55-kDa cementum-
attachment protein (196, 263). The presence of other
bioactive proteins, such as enamel-like proteins (235,
234), osteonectin/SPARC (201), and mitogenic factors
(157, 269), have also been reported in the cementum.
In addition to these proteins, cementoblasts synthes-
ize and secrete several glycosaminoglycans (such as
chondroitin-4-sulfate, chondroitin-6-sulfate and der-
matan sulfate, and collagen fibrils), which are present
in the cemento–dentinal junction (88, 264, 265).
It has been suggested that cementoblasts exhibit
an osteoblast-like, rather than an odontoblast-
like, phenotype (25). Odontoblast, osteoblast and
cementoblast cells express several matrix proteins,
such as osteopontin, bone sialoprotein (BSP), osteo-
nectin, osteocalcin, matrix Gla protein (208) and den-
tin-matrix-protein 1 (DMP-1) (106). The presence of
osteocalcin in cementum is more controversial.
Bronckers et al. (25), using immunohistochemistry,
reported the presence of osteocalcin on the cellular
intrinsic fiber cementum (CIFC) and associated
cementoblasts (mature), but not in the acellular
cementum and its associated cementoblasts. Tenorio
et al. (246) reported the presence of osteocalcin in
acellular extrinsic fiber cementum (AEFC) but not in
the associated cementoblasts, while CIFC and associ-
ated cementoblasts stainedweakly. Bosshardt &Nanci
(20) used two different antibodies (OC1 and OC2),
which gave different results: OC1 showed reactivity
with acellular cementum, while OC2 was negative.
Similarly, the presence of DMP-1 has been associated
with acellular cementum (275) and cementocytes, but
not with cementoblasts (255). It has been suggested
that acellular cementum is a unique tissue, while cel-
lular cementum and bone share some similarities,
although there are still morphological, functional and
biochemical differences between the two tissues (19).
The presence of cementum-specific proteins
remains questionable, although some putative
199
Regeneration of periodontal tissues: cementogenesis revisited
cementum-specific proteins have been invoked: a
55-kDa CAP (263); a mitogenic factor (167); and a
72-kDa protein, CEM-1 (235). However, as the char-
acterization and the sole expression by cementoblasts
of these proteins have not been determined, the
possible existence of cementum-specific proteins
remains unknown. It has been reported that
cementoblasts and cementocytes produce high levels
of the GLUT-1 monosaccharide transporter, while
osteoblasts or osteocytes do not express this protein.
These data suggest that GLUT-1 may play a role in
cementogenesis and could serve as a biomarker to
differentiate between cells of cementoblastic and
osteoblastic lineage (124). However, the observed
differences in GLUT-1 are quantitative, and GLUT-1 is
present in many different cell types. Recently, we
reported the isolation of a cementoblastoma-derived
protein, CP-23, that is expressed by cementoblasts and
some precursor cells present in the periodontal liga-
ment, butnot byosteoblasts. The functionof theCP-23
protein is currently unknown; however, given its
nuclear location, it may be required for cementoblast
differentiation and may be used as a marker for
cementoblast cells (3). The CP-23 protein is also ex-
pressed by Hertwig’s epithelial root sheath cells (275).
Based on our current knowledge of the develop-
ment of periodontal tissues, several strategies exist
for targeting regenerative therapy, ranging from
inducing their own �regenerative� mechanisms using
molecular approaches, or utilizing cells to repopulate
and recapitulate the developmental process.
Strategies for periodontalregeneration/repair
The process of periodontal tissue regeneration starts
at the moment of tissue damage by means of growth
factors and cytokines released by the damaged con-
nective tissue and inflammatory cells. It is well
accepted that in order to improve periodontal healing,
root planing or root conditioning is a necessary
antecedent to mesenchymal cell migration and
attachment onto the exposed root surface. Acid
treatment, in particular with citric acid, has been
found to widen the orifices of dentinal tubules,
thereby accelerating cementogenesis and increasing
cementum apposition and connective tissue attach-
ment. However, a systematic review performed by
Mariotti (145) suggested that the use of citric acid,
tetracycline or EDTA to modify the root surface pro-
vides no clinical significant benefit for regeneration in
patients with chronic periodontitis. Conversely, when
periodontal ligament cells are removed from the ce-
mentum or are unable to regenerate, bone tissue in-
vades the periodontal ligament space and establishes
a direct connection between the tooth and the wall of
the alveolar socket, resulting in ankylosis. The ankyl-
otic, nonflexible type of tooth support can lead to loss
of function and resorption of the tooth root (13).
Can guided tissue regeneration and bonegrafting regenerate cementum?
Nyman et al. (174), using Millipore� membranes,
introduced the concept of a membrane barrier, which
excludes the apical migration of gingival epithelial
cells and provides an isolated space for the inwards
migration of periodontal ligament cells, osteoblasts
and cementoblasts. Guided tissue regeneration was
successfully used to aid in the regeneration of lost
periodontal tissues caused by periodontitis (67). The
first guided tissue regeneration membranes were
nonabsorbable and made of polytetrafluoroethylene,
such as Gore-Tex�. Studies on experimentally
induced periodontal defects in monkeys suggested
that guided tissue regeneration was capable of
inducing the formation of new bone and cementum
(4). The second generation of guided tissue regener-
ation used absorbable membranes made of collagen
or polylactic and citric acid (28, 159), which elimin-
ated the need for surgical membrane retrieval (66).
Recent systematic reviews indicate that, in the
treatment of intrabony and furcation defects, guided
tissue regeneration is more effective than open flap
debridement. Various barrier types yielded no sys-
tematic difference in clinical outcome, but barrier
types could explain some heterogeneity in the results.
Overall, guided tissue regeneration is consistently
more effective than open flap debridement in the
gain of clinical attachment and reduction of probing
depth in the treatment of intrabony and furcation
defects (99, 163). The use of grafting material in
combination with guided tissue regeneration seems
to improve clinical outcomes for furcation, but not
for intrabony defects, when compared with the use of
barrier membranes alone. It has also been questioned
whether guided tissue regeneration produces true
cementum regeneration or only cemental repair. The
newly formed cementum has been characterized as a
cellular cementum that is usually poorly attached to
the dentin surface (125). It is suggested that perio-
dontal healing with guided tissue regeneration ther-
apy occurs in two stages. The first stage comprises an
initial healing phase with the formation of a blood
clot, transient root resorption/demineralization,
200
Zeichner-David
deposition of acellular cementum on the root surface
and formation of connective tissue. The second
phase comprises a remodeling process, which will
result in a regenerated cementum similar to pristine
cementum as maturation proceeds over time (69). In
conclusion, several clinical studies have demonstra-
ted that guided tissue regeneration is a successful
treatment modality for periodontal reconstructive
surgery and it has become an accepted procedure in
most periodontal practices, either by itself or in
combination with other treatment modalities.
Autologous bone grafts to repair periodontal osse-
ous defects have been used for many years and dif-
ferent approaches have been the subject of several
reviews (165, 209). Bone repair can also be achieved
using ceramic materials such as Bioglass, which is a
bone-bonding bioactive material that has been widely
used for bone healing (110). Studies in monkeys sug-
gested that PerioGlas� (synthetic bone particulate)
can achieve superior bone repair and cementum
regeneration and retard epithelial down-growth
compared with other, similar materials (50, 109).
Additionally, these materials can be used as scaffolds
or todeliver otherbioactivemolecules to enhance their
function. The use of bone grafts, powders or ceramics
is quite prevalent in many dental practices. A recent
systematic review on the efficacy of bone replacement
grafts compared with other interventions in the treat-
ment of periodontal osseous defects was performed by
Reynolds et al. (202). Meta-analysis indicated that for
the treatment of intrabony defects, bone grafts are
effective in reducing crestal bone loss, increasing bone
level, increasing clinical attachment level, and redu-
cing probing depth compared with open flap debri-
dement procedures. Histological studies showed that
demineralized freeze-driedboneallografts support the
formation of a new attachment apparatus in intrabony
defects; however, the available data indicate that
alloplastic grafts support periodontal repair rather
than regeneration, and that the best treatment is a
combination of bone grafts with barrier membranes.
Nevertheless, these strategies are directed mainly to
enhance alveolar bone and periodontal ligament
repair and have the problems that they do not address
cementogenesis and therefore do not completely
regenerate the architecture of the original periodon-
tium.
Molecular approaches for cementumregeneration
Advances in our knowledge of developmental bio-
logy, and of the growth factors that initiate and
regulate tooth development and tissue repair, sug-
gests the use of some of these factors for periodon-
tium regeneration (37, 61, 68, 71, 118, 116, 117, 128,
170). Some attachment proteins, such as fibronectin
(29, 206, 262) or CAP (156, 196), are able to enhance
fibroblast migration, attachment and orientation of
the connective tissue to the root surface. New
strategies, utilizing growth factors to induce cell
migration, proliferation and differentiation, were
developed to repopulate the damaged periodontal
tissues with periodontal ligament cells (32, 247). It is
believed that growth factors play important roles in
modulating the proliferation and/or migration and/
or differentiation of structural cells in the periodon-
tium (58, 86, 97, 197, 230). It is suggested that growth
factor molecules are produced during cementum
formation and then stored in the mature cementum
matrix with the potential to induce periodontal repair
or regeneration when needed (236). Large-scale pro-
duction of recombinant growth factors has facilitated
in vitro and in vivo studies to determine the efficacy
of growth factors in periodontal tissue regeneration.
Amongst the growth factors currently being used
are platelet-derived growth factor, insulin-like growth
factor (36, 63, 92, 138, 188, 210), transforming growth
factor-b1 (146), basic fibroblast growth factor (213),
dexamethasone (211) and BMPs (121, 205, 211).
However, problems in applying these growth factors
for periodontal repair include the nonspecific activity
of some factors on different cell lineages in time and
space, and the rapid loss of growth factors applied
topically (13, 138).
It has been shown that both platelet-derived
growth factor and insulin-like growth factor-1 can
stimulate the proliferation and chemotaxis of perio-
dontal ligament cells, and that the combination of
platelet-derived growth factor and insulin-like growth
factor-1 can further increase the mitogenic effect (23,
44, 175). In addition to the mitogenic activity, plate-
let-derived growth factor also appears to stimulate
collagen synthesis in periodontal ligament cells (146).
Furthermore, dexamethasone has been shown to
exert the same effect as insulin-like growth factor-1
on periodontal ligament fibroblasts, gingival fibro-
blasts and pulp fibroblasts, and may substitute for
insulin-like growth factor-1 in the platelet-derived
growth factor stimulation of cell proliferation (210).
In addition to the previously described effects,
platelet-derived growth factor has the capacity to
significantly negate and reverse the inhibitory effects
of lipopolysaccharide on the proliferation of human
gingival fibroblasts. Lipopolysaccharide from a vari-
ety of gram-negative bacteria is known to inhibit
201
Regeneration of periodontal tissues: cementogenesis revisited
gingival fibroblast proliferation and synthesizing
activity, has been implicated in periodontal inflam-
mation and may also be responsible for delayed
wound healing following periodontal therapy (12).
In vivo studies using the beagle dog (natural perio-
dontal disease) and the nonhuman primate (ligature-
induced attachment loss) models showed that the
application of platelet-derived growth factor/insulin-
like growth factor-1 resulted in significant amounts of
new bone and cementum formation (138, 210).
Treatment with insulin-like growth factor-1 alone did
not significantly alter healing compared with controls,
while treatment with platelet-derived growth factor
alone showed significant regeneration of attachment.
Although there are differences in the response to
platelet-derived growth factor/insulin-like growth
factor-1, depending on which animal model is used
(the osseous response in dogs appears to be greater
than that of the nonhuman primate, while new
attachment formation appears to be greater in the
nonhuman primate than in the dog), there is consis-
tency in promoting periodontal regeneration (63, 64).
Rutherford et al. (211) showed that platelet-derived
growth factor and dexamethasone, combined with a
collagen carrier matrix, induced regeneration of the
periodontium inmonkeys. It has also been shown that
the combination of platelet-derived growth factor and
guided tissue regeneration work better than either of
the two modalities alone (36, 188).
Clinical trials in humans using platelet-derived
growth factor/insulin-like growth factor to treat per-
iodontal osseous defects showed that only high doses
of these factors gave rise to a statistically significant
increase in alveolar bone formation (92). When
platelet-derived growth factor was used in combina-
tion with bone allografts to treat Class II furcations
and interproximal intrabony defects, histological
evaluation showed regeneration of new alveolar
bone, cementum, and periodontal ligament (30, 171).
Platelet-rich plasma is a fraction of plasma that
contains platelet-derived growth factor and trans-
forming growth factor-b (180). An alternative to the
use of recombinant growth factors is the use of a
platelet gel in combination with demineralized
freeze-dried bone allografts (5, 43).
The limitations of topical protein delivery to peri-
odontal osseous defects include transient biological
activity and bioavailability of platelet-derived growth
factor at the wound site. To overcome these limita-
tions, studies have used genetic engineering to
transduce cells derived from the periodontium, using
adenovirus carrying the platelet-derived growth fac-
tor gene to promote sustained release and ensure
biological activity (7, 6, 65). The potential use of gene
therapy in vivo to stimulate periodontal tissue
regeneration has been studied in large tooth-associ-
ated alveolar bony defects in rats. The results showed
that the direct gene transfer of platelet-derived
growth factor-B stimulates the regeneration of
alveolar bone and cementum (104).
As stated above, some members of the BMPs are
normally expressed during the development of the
periodontium, such as BMP-3 and BMP-7/OP-1,
which have been localized immunologically in
alveolar bone, cementum, and periodontal ligament,
whereas BMP-2 was only localized in the alveolar
bone (249, 266). Although the exact role of BMPs in
the development of the periodontium has not yet
been determined, these proteins are good candidates
for stimulating periodontal regeneration because of
their ability to promote not only osteogenesis but
also cementogenesis. The expected role of BMPs in
stimulating intramembranous bone formation with-
out an endochondral intermediate may provide
greater osteogenic potential than autogenous bone or
other bone substitutes (121, 118, 119, 170, 205, 240).
Studies indicate that recombinant BMP-2 exerts no
effect on the growth and differentiation of human
periodontal ligament cells in vitro; however, BMP-2
stimulates alkaline phosphatase activity and para-
thyroid hormone-dependent 3¢,5¢-cyclic adenosine
monophosphate (cAMP) accumulation, which are
early markers of osteoblast differentiation. Never-
theless, BMP-2 produced no mature osteoblasts, as
measured by expression of osteocalcin, and also
inhibited 1,25(OH)2D3-induced osteocalcin synthesis
in these cells (123). In vitro studies using mouse-de-
rived dental follicle and periodontal ligament cells
suggest that BMP-2 induced dental follicle cells to
differentiate towards a cementoblast/osteoblast phe-
notype but had no effect on periodontal ligament cells
(278). Paradoxally, BMP-2 was found to inhibit ce-
mentoblast cell mineralization in vitro by decreasing
the expression of BSP and collagen type 1 (279). In
studies of BMP-2 on early wound healing in a rat
model of periodontal regeneration, the connective
tissue attachment was found to be similar in animals
receiving BMP-2 and in controls. However, BMP-2
induced bone formation at some distance from the
defect, which indicates the need for a suitable delivery
system to maintain the BMP-2 at the site of implan-
tation (120). Other studies suggest that the effects of
BMPs may be influenced by certain factors, such as
root surface conditioning, delivery systems, mastica-
tory forces, etc., and that BMP-2 stimulates the pro-
liferation and migration of cells from the adjacent
202
Zeichner-David
periodontal ligament into the wounded area, pro-
moting new cementum formation (119).
The expression of both BMP-2 and BMP-7 during
periodontal tissue morphogenesis suggests that
optimal therapeutic regeneration may require the
combined use of the two BMPs. BMP-7-treated molar
furcation defects in baboons resulted in substantial
cementogenesis, while BMP-2 showed limited
cementum formation but greater amounts of miner-
alized bone and osteoid; however, the combined
application did not enhance alveolar bone regener-
ation or new attachment formation over and above
that obtained by separate applications of the two
BMPs (207). Recently, it was shown that the appli-
cation of a synthetic BMP-6 polypeptide to a perio-
dontal fenestration defect in rats resulted in
increased formation of new bone and cementum
(93). Perhaps the use of other members of the BMP
family, such as growth and differentiation factor-5,
)6, and )7, might provide better and more complete
regenerative outcomes. These factors have been
detected during the process of periodontal develop-
ment at the surfaces of alveolar bone, cementum and
periodontal ligament fiber bundles (223).
Limitations for the regular use of BMPs are the
need for high doses, non-specific activity on different
cell lineages in time and space, and the rapid loss of
topically applied growth factors (13, 138). Some of
these problems can be overcome by the use of gene
transfer technology. Jin et al. (103) used adenoviruses
containing BMP-7 to transduce dermal fibroblasts
that were then used to treat mandibular alveolar
bone defects in a rat wound repair model. Their
results showed chrondrogenesis, with subsequent
osteogenesis, cementogenesis and bridging of the
periodontal bone defects, suggesting that this genetic
engineering approach may be useful in alveolar bone
regeneration. A recent literature review (62) conclu-
ded that although promising, there were insufficient
data at the present time to conduct a meta-analysis
on the effect of growth factors for periodontal repair,
and pointed to the need for more clinical trials.
Do enamel-associated proteinsregenerate cementum?
Based on the presence of enamel proteins in acellular
cementum (133, 235, 182, 233), it was thought that
these proteins may play a role in the repair/regen-
eration of periodontal tissues destroyed by perio-
dontal disease (78). This idea was tested by adding
enamel proteins or purified enamel matrix derivative
to surgically produced periodontal defects in mon-
keys, followed by histological analysis that showed
almost complete regeneration of acellular cementum,
firmly attached to the dentin and with collagenous
fibers extending towards newly formed alveolar bone
(79). These studies resulted in a new therapeutic
preparation to treat periodontal disease, consisting of
hydrophobic enamel matrix proteins extracted from
porcine developing enamel, which has been marke-
ted by Biora, Inc., under the name of Emdogain�. In
the past 8 years, the use of enamel proteins for
inducing the formation of cementum, bone and
dentin has generated numerous in vivo and in vitro
studies, as well as clinical trials, resulting in almost
300 publications. In vitro studies, animal studies and
clinical trials are all being conducted simultaneously
(60, 70, 83, 154).
In vitro studies, using periodontal-associated cells
such as periodontal ligament fibroblasts, osteoblasts,
cementoblasts, gingival fibroblasts, gingival epithelial
cells, etc., have been conducted in an attempt to
understand the molecular and cellular mechanisms
involved in the process of enamel matrix derivative-
induced tissue regeneration. In order for enamel
matrix derivative to regenerate periodontal tissues, it
will need to exert an effect on proliferation, migra-
tion, attachment and/or differentiation of the sur-
rounding periodontal cells, and most studies have
measured these parameters, as shown in Table 1.
Few studies have tested the effect of enamel matrix
derivative on cell migration, but available data sug-
gest an increased migration of periodontal ligament
cells, osteoblasts, gingival fibroblasts and dermal
fibroblasts in response to enamel matrix derivative,
with the exception of one study that found no effect
on periodontal ligament cells (184). Most studies on
the effect of enamel matrix derivative on cell
attachment, which generally included periodontal
ligament cells, found an increase in cell attachment
(184). However, one study found the enamel matrix
derivative to have no effect on cell attachment of
gingival fibroblasts (256). A number of studies, which
measured the effect of enamel matrix derivative on
cell proliferation, have found an increase in cell
proliferation in the presence of enamel matrix
derivative. However, the proliferative effect was not
found in two studies using periodontal ligament cells
(41, 256), in two studies using osteoblast cell lines
(215, 268) and in one study using gingival fibroblasts
(256). Several studies found an inhibition of cell
proliferation when epithelial cells were used (112,
139, 273). These data may explain the clinical
observation that application of enamel matrix
derivative suppresses the down-growth of junctional
203
Regeneration of periodontal tissues: cementogenesis revisited
Table
1.In
vitro
studiesontheeffectofenamelprotein
derivativeoncells
Ce
lls
Sp
ec
ies
Mig
rati
on
Att
ac
hm
en
tP
roli
fera
tio
nD
iffe
ren
tia
tio
nM
ine
rali
zati
on
Re
fere
nc
e
Periodontalligamentcells
Human
ND
ND
ND
APincrease
–osteoblast
Yes
(166)
Human
Noeffect
Noeffect
Yes–increase
No(TypeIcol)
ND
(184)
Rat(primary)
ND
ND
Yes–decrease
No–Col,AP
(95)
Human(primary)
ND
ND
Yes–increase
Yes–less
AP–cementoblast
ND
(33)
Human(primary)
Yes
ND
Yes–increase
ND
ND
(203)
Human(primary)
ND
Yes
Nodifference
ND
ND
(41)
Human(primary)
ND
ND
Yes–increase
Yes,
increase
IGF-1
andTGF-b1.
Noeffectonbonephenotype
ND
(178)
Hu(primary)
ND
ND
ND
Increase
matrix
(versican,biglycan,
decorin,hyaluronan
ND
(73)
Hu(primary)
ND
Yes
Noeffect
Increase
APandTGF-b1
ND
(256)
Hu(primary)
Yes
ND
Yes–increase
ND
ND
(89)
Hu(primary)
ND
Yes
Yes–increase
Increase
cAMP,TGF-b1,IL-6,PDGF-A
BND
(139)
P(primary)
ND
Yes
Yes–increase
Increase
OPN
ND
(204)
Mo(cellline)**
ND
Yes
Yes–increase
InhibitsColI,denovoexp
ressionBSP
andOCN,increase
BMP2
ND
(273)
Mo(cellline)�
ND
Yes
Yes–increase
InhibitsColI,denovoOCN
andBMP3
ND
(273)
Osteoblasts
Hu(ROS17/2.8)
ND
ND
ND
BSPincrease
ND
(227)
Hu(primary)
ND
ND
Yes–increase
More
FGF2andCOX2;less
APandMMP1
ND
(161)
Mo(ST2)
ND
ND
Noeffect
Yes–AP
ND
(268)
Mo(K
USA/A
1)
ND
ND
Yes–increase
Yes–AP,Col,OPN,TGF-b1,OCN
andMMPS
Yes-
more
(268)
Mo(primary)
ND
ND
Yes–increase
ND
ND
(101)
Mo(primary)
ND
ND
ND
Increase
Col,IL-6
andPGHS-2;
noeffectonOCN
andIG
F-1
ND
(102)
Mo(M
C3T3-E1)
ND
ND
Yes–increase
Increase
OPN
andless
OCN
(254)
Hu(2T9pre-osteoblasts)
ND
ND
Yes–increase
Noeffect
ND
(215)
Hu(M
G63osteoblast
like)
ND
ND
Yes–decrease
Yes,
increase
AP,OCN,TGB1
ND
(215)
204
Zeichner-David
Table
1.Continued
Ce
lls
Sp
ec
ies
Mig
rati
on
Att
ac
hm
en
tP
roli
fera
tio
nD
iffe
ren
tia
tio
nM
ine
rali
zati
on
Re
fere
nc
e
Hu(primary)
ND
ND
Yes–increase
Yes,
increase
AP,OCN,TGB1
ND
(215)
Hu(M
G63)
Yes
ND
Yes–increase
ND
ND
(89)
P(primary)
ND
Yes
Yes–increase
Increase
OPN
ND
(204)
Hu(Ros17/28)*
ND
ND
ND
BSPincrease
ND
(228)
Gingivalfibroblast
cells
Rat
ND
ND
Yes–double
Faster–osteogenic
Yes–more
(115)
Hu(primary)
Yes
ND
Yes–increase
ND
ND
(203)
Hu(primary)
ND
ND
ND
Increase
matrix
(versican,biglycan,
decorin,hyaluronan
ND
(73)
Hu(primary)
ND
Noeffect
Noeffect
Increase
APandTGF-b1
ND
(256)
Hu(primary)
Yes
ND
Yes–increase
ND
ND
(89)
Rat
ND
ND
Yes–increase
More
ECM
No
(115)
Rat
ND
ND
Nodifference
ND
ND
(72)
P(primary)
ND
Yes
Yes–increase
Increase
OPN
ND
(204)
Dentalfollicle
Mo(SV40)
ND
ND
Yes–increase
More
OPN,Less
OCN
Inhibits
(74)
Cementoblasts
Mo(SV40)
ND
ND
Yes–increase
Decrease
Ocn
Inhibits
(254)
Mo(O
CCM-30)*
ND
ND
ND
Decrease
BSP
Inhibits
(258)
Mo(O
CCM-30)N
D�
ND
Noeffect
Decrease
OCN,increase
OPN
andOPG
Inhibits
(17)
Fibroblasts
Mo(L929)
ND
ND
Nodifference
ND
ND
(72)
Rabbit
Yes–vascularendothelium.Growth
factors
(160)
Human(primary)
Yes
ND
Yes–increase
ND
ND
(203)
Mesenchymalstem
cells
Hu(C
2C12)
ND
ND
ND
Yes–increase
AP.Osteoblast
phenotype
ND
(177)
Epithelialcells
Hu(H
ELA)
ND
ND
Inhibited
Increase
cAMPandPDGF-A
BND
(113)
Hu(SCC25)
ND
ND
Inhibited
Increase
p21WAF1/cip1;decrease
CK-18
ND
(113,112,114)
ERM
P(primary)
ND
Yes
Yes–increase
Increase
OPN
ND
(204)
Endothelialcells
Hu(H
UVEC)
Yes–increase
ND
Noeffect
ND
ND
(271)
AP,alkalinephosp
hatase;BMP,bonematrix
protein;BSP,bonesialoprotein;Col,collagen;Hu,human;IG
F-1,insu
lin-likegrowth
factor;IL-6,interleukin-6;MMPS,matrix
metalloproteinases;Mo,mouse;ND,notdeterm
ined;
OCN,osteocalcin;OPN,osteopontin;OPG,osteoprotegerin;P,pig;PDGF-A
B,plateletderivedgrowth
factorAB;PGHS-2,prostaglandin
G/H
synthase
2;TGF-b1,transform
inggrowth
factorb-1.
*Mouse
recombinantamelogenin.�Mouse
recombinantameloblastin.�Mouse
leucinerichamelogenin
peptide(LRAP).
205
Regeneration of periodontal tissues: cementogenesis revisited
epithelium onto dental root surfaces, a process that
frequently interferes with the formation of new con-
nective tissue attachments (79, 78).
The majority of available in vitro studies have
analyzed the effect of enamel matrix derivative on
gene expression and differentiation, and most of
these studies found either an increased or a de-
creased expression of certain transcription and
growth factors, extracellular matrix proteins or min-
eralization-associated proteins in the cells tested.
Where mineralization was measured, it was found
that enamel matrix derivative induced mineralization
of periodontal ligament cells (166), increased miner-
alization of osteoblasts (268) and gingival fibroblasts
(115), decreased mineralization of cementoblast cells
(254) and inhibited the mineralization of dental fol-
licle cells (74). Differences in results amongst studies
can be explained by differences in sources and con-
centrations of enamel matrix derivative and in the
cell preparations used. Most studies employed pri-
mary cell cultures derived from different patients,
which probably contained mixed populations of a
variety of cells present in the periodontium. Never-
theless, taken together, these studies suggest that
enamel matrix derivative can act as a multipurpose
growth factor capable of stimulating the proliferation
of mesenchymal cells while inhibiting the cell divi-
sion of epithelial cells, and can stimulate attachment
and phenotypical changes in some cells, while
inhibiting matrix production in others.
Given the widespread use of Emdogain�, and the
fact that it is made from an extract of enamel pro-
teins, it is important to identify the actual protein
responsible for its function. Studies by Maycock et al.
(148) found that, in addition to amelogenin, Emdo-
gain� contains metalloproteases and serine pro-
teases. Studies by Kawase et al. (114) demonstrated
that porcine enamel matrix derivative contains
transforming growth factor-b1 (or a transforming
growth factor-b-like substance), and that the action
of enamel matrix derivative is mediated by the smad-
2 signaling pathway. In addition, a neutralizing
anti-transforming growth factor-b immunoglobulin
blocked the action of enamel matrix derivative on
epithelial cells, although it failed to block completely
enamel matrix derivative-induced fibroblastic prolif-
eration, suggesting the presence of more than one
growth factor. Iwata et al. (98) isolated the inductive
activity of enamel matrix derivative by using chro-
matography and characterized it as being BMP-2 and
BMP-4 using specific antibodies. Furthermore, in the
presence of noggin (an inhibitor of BMPs), enamel
matrix derivative lost its inductive activity, indicating
that BMPs are the molecules responsible for enamel
matrix derivative activity. Although these studies
suggest that the action of Emdogain� is a result of
the presence of contaminating growth factors, other
studies have shown that pure recombinant enamel
proteins indeed have activity as inducers. The results
obtained in our laboratory indicate that mouse
recombinant amelogenin can increase attachment
and proliferation of mouse periodontal ligament cells
in vitro (272, 273). Furthermore, a post-translational
modified recombinant ameloblastin, another enamel-
associated protein, had an effect similar to that of
amelogenin on periodontal ligament cells. Both
recombinant amelogenin and ameloblastin can
change the phenotype expressed by periodontal
ligament cells by inhibiting the expression of colla-
gen type I and inducing de novo expression of
osteocalcin. Amelogenin also induced the expression
of bone sialoprotein and BMP-2, while ameloblastin
induced the de novo expression of BMP-3 (273).
These results indicate that both enamel-associated
proteins have a modulatory effect on the expression
of BMPs, suggesting that perhaps these proteins exert
their signaling effect by means of BMPs. Recombin-
ant mouse amelogenin improved osteoblast adhe-
sion (90), and increased the expression of bone
sialoprotein and decreased the formation of miner-
alized nodules in cementoblasts (258). A leucine-rich
amelogenin peptide, which exhibited no effect on
cell proliferation, down-regulated osteocalcin and
up-regulated osteopontin in a dose- and time-
dependent manner, and inhibited the capacity to
form mineral nodules (17). Taken together, these
reports point towards a growth factor activity for
enamel proteins that may be of importance in
periodontal tissue regeneration.
Several clinical trials have shown an increase in
periodontal attachment and bone formation in indi-
viduals treated with Emdogain� (54, 85, 87, 154, 179,
200, 217, 216, 218, 219, 277). However, in many of
these studies, the results were no better than those
obtained with other previously established treat-
ments, such as guided tissue regeneration, which
yields better outcomes in the management of deep
intrabony periodontal defects (84, 187, 218, 221, 231).
Histological studies revealed that treatment with
Emdogain� is unpredictable, resulting in the forma-
tion of cellular cementum rather than acellular
cementum, and this cementum was only partially
attached to the root surface, similar to the cementum
formed with the use of guided tissue regeneration.
Furthermore, more bone regeneration occurred by
using a guided tissue regeneration procedure than
206
Zeichner-David
Emdogain� (216, 219, 218). Other studies showed no
evidence of improvement in radiographic bone level,
and surgical re-entry found new tissue with a rubbery
consistency and that was not mineralized (189, 190).
Experiments in rats, using a wounded rat periodon-
tium model followed by immunohistochemical
analysis, showed that Emdogain� does not affect the
expression of differentiation markers or bone matrix
protein synthesis in the repopulation response of
wounded rat molar periodontium (35).
Systematic studies, using literature reviews and
meta-analysis, suggest that treatment with enamel
matrix derivative results in significant variations in
clinical outcomes (107). Although Emdogain� is able
to significantly improve probing attachment levels
and pocket depth reduction, some studies found no
evidence of clinically important differences between
guided tissue regeneration and Emdogain� (47, 62)
and reported that guided tissue regeneration is more
predictable for cementum and bone regeneration
(257). Although animal histological studies with sur-
gically created defects suggest that enamel matrix
derivative induces the formation of acellular cemen-
tum and promotes attachment of the supporting
periodontal tissues, human histological studies have
questioned both the consistency of the histological
outcomes and the ability of enamel matrix derivative
to predictably stimulate the formation of acellular
cementum (107). It appears that following treatment
with enamel matrix derivative, a bone-like tissue
resembling cellular intrinsic fibrous cementum is
formed (22).
Despite the mixed results obtained from both
in vitro and in vivo studies, new applications of
Emdogain� are continuously being reported. Some
studies suggest that it has the ability to induce the
formation of reparative dentin in pulpotomized teeth
(94, 96, 168, 169). It is being used to coat titanium
implants with mixed results; one study suggests that
there is enhanced formation of trabecular bone (229)
while the other found no effect (53). It has also been
suggested that enamel matrix derivative can combat
bacteria in postsurgical periodontal wounds, which
otherwise could hamper wound healing and reduce
the outcome of regenerative procedures (8, 172, 220,
237). More recently, an acceleration of skin wound
14 days
PLF PL-7 DPM
Control ControlHERS-CM HERS-CM Control HERS-CM
21 days
28 days
35 days
Fig. 2. Effect of Hertwig’s epithelial root sheath-condi-
tioned media (HERS-CM) on periodontium-associated cell
mineralization. HERS-CM was prepared by growing the
cells in Dulbecco’s modified Eagle’s minimal essential
medium (DMEM) supplemented with 10% fetal calf serum
(FCS) and 100 U/ml of penicillin/streptomycin. Cells were
incubated at 39.5�C in a humidified atmosphere of 95%
air and 5% CO2 for 7 days, after which the media were
collected, the protein concentration determined and then
lyophilized. Periodontal ligament fibroblasts (PLF), ce-
mentoblasts (PL-7) and dental papillae mesenchyme
fibroblasts (DPM) were prepared from Immortomouse
(275). Cells were grown in differentiation conditions
(DMEM supplemented with 10% FCS, 100 U/ml of peni-
cillin/streptomycin, 50 mg/ml of ascorbic acid and 2 mM
sodium b-glycerophosphate), with or without (controls)
100 lg of HERS-CM proteins. At different time-points of
culture, cells were fixed with 70% methanol and 30%
acetic acid and stained with Von Kossa to determine
mineralization.
207
Regeneration of periodontal tissues: cementogenesis revisited
healing in the presence of enamel matrix derivative
was reported (160).
Cellular tissue engineering forcementum regeneration
It has long been recognized that a recolonization of
periodontal ligament cells onto the root surface is
necessary for periodontal ligament regeneration (129,
174). One therapeutic approach proposed the removal
of autologous cells from the patient’s periodontal
ligament, culture of the cells in vitro, to place them
back onto the exposed root coated with chemo-
attractant factors, and then to cover the area with an
artificial basement membrane (247). A pilot study was
carried out with four patients, using hydroxyapatite as
a vehicle for cell delivery. After 6 months, the treated
patients exhibited greater pocket reduction and clin-
ical attachment gain, and less gingival recession, than
control patients; however, both groups showed good
fill of the osseous defects studied (48, 49, 91).
Lekic et al. (130) tracked the fate and differenti-
ation of rat periodontal cells and bone marrow cells
transplanted into periodontal wounds in rats using
cells constitutively expressing b-galactosidase as a
marker. Labeled cells were localized in the perio-
dontal ligament and regenerating alveolar bone and it
was suggested that, following a cyclical process of
growth and development, both cell types were able to
differentiate into periodontal ligament fibroblasts,
osteoblasts and cementoblasts, and to contribute to
periodontal regeneration (131). Regeneration of
cementum, periodontal ligament and alveolar bone
has also been observed using auto-transplantation of
bone marrow mesenchymal stem cells into perio-
dontal osseous defects in dogs (111). Similar results
have been observed after the application of perio-
dontal ligament cell sheets (2).
The ability of cementoblasts and dental follicle
cells to promote periodontal regeneration in a rodent
periodontal fenestration model was analyzed recently
(280). The results indicated that cementoblast-trea-
ted and carrier alone-treated defects showed com-
plete bone bridging and periodontal ligament
formation; however, no new cementum was formed
along the root surface in either group. Puzzling,
however, was the fact that no repair, or even osteo-
genesis, was seen within dental follicle cell-treated
defects, even though these cells are believed to be
precursors of cementoblasts and to be responsible for
alveolar bone formation.
As our laboratory has established immortal cell
lines for the Hertwig’s epithelial root sheath (275) and
the Epithelial Rest of Malassez cells, we are exploring
the ability of these cells, or their secreted products, to
induce periodontal ligament cells to differentiate into
cementoblasts in vitro. When periodontal ligament
cells, which do not produce a mineralized extracel-
lular matrix, are grown in the presence of Hertwig’s
epithelial root sheath conditioned media (HERS-CM),
these cells produce a mineralized extracellular mat-
rix, as determined by a positive Von-Kossa staining
Effect of HERS-CM on PLFcell differentiation
P
BSP
OCN
OSN
OPN
AP
BMP4
Col1
Actin
21d 21d + HERS
Fig. 3. Effect of Hertwig’s epithelial root sheath-condi-
tioned media (HERS-CM) on the phenotype of periodontal
ligament cells. HERS-CM was prepared as previously
described. Periodontal ligament cells were grown under
proliferation (P) conditions (in the presence of interferon-
c at 33�C) or differentiation conditions [Dulbecco’s
modified Eagle’s minimal essential medium (DMEM)
supplemented with 10% fetal calf serum (FCS), 100 U/ml
of penicillin/streptomycin, 50 mg/ml of ascorbic acid and
2 mM sodium b-glycerophosphate] with or without (con-
trols) 100 lg of HERS-CM proteins. Cells were collected
after 21 days in culture (media were changed every other
day), the media were removed, cells were rinsed in
phosphate-buffered saline (PBS) and total RNA was
extracted for determination of phenotype by using reverse
transcription–polymerase chain reaction (RT–PCR). AP,
alkaline phosphatase; BMP-4, bone morphogenetic pro-
tein-4; BSP, bone sialoprotein; Col1, collagen type I; OCN,
osteocalcin; OPN, osteopontin; OSN, osteonectin.
208
Zeichner-David
(Fig. 2). This effect is specific for periodontal liga-
ment cells because other types of fibroblasts, such as
those derived from the dental pulp, do not produce a
mineralized extracellular matrix, even in the presence
of HERS-CM. When cementoblast cells, capable of
producing a mineralized extracellular matrix, were
grown in the presence of HERS-CM, an acceleration
in the formation of mineral was detected. Analysis of
the phenotype at the molecular level indicated a
de novo induction of the expression of bone sialo-
protein and osteocalcin, two markers of mineraliza-
tion (Fig. 3). These results support the concept that,
during root development, the secreted products of
the Hertwig’s epithelial root sheath induce adjacent
cells of the periodontal ligament to differentiate and
produce new cementum. However, whether these
cells differentiate into cementoblasts or osteoblasts
awaits further in vivo experiments.
Conclusions
It is obvious that major progress has occurred in the
world of biology, medicine and dentistry in the past
30 years, and the management of periodontal disease
has benefited from these advances. New knowledge
about the etiology and pathogenesis of periodontitis,
the relationship of the disease to systemic problems,
and advances in genetics, molecular biology, cell
biology and biomaterials, have opened the door for
new regenerative techniques based upon the tissue
engineering approach. Treatment of periodontal
disease has evolved from just fighting bacteria to a
combined effort to eliminate the offending microor-
ganisms, to arrest the progression of tissue damage
and to regenerate lost tissues. Although some of the
regenerative techniques have been available for sev-
eral years, and some have shown promising results,
none of the techniques are without problems and
none have proven to be 100% effective. Many of the
regenerative approaches reviewed in this article are
still under assessment and further research is needed
to develop cell-based tissue strategies, perhaps using
stem cells and biomaterials for delivery of these cells.
New scaffold materials, which are being developed,
are also needed to address some of the delivery issues
(164). What may be concluded from the current sta-
tus of periodontal regeneration is that, as many
investigators have previously stated, there is not
going to be one magic solution that can be used to
treat all periodontal patients, but rather a combina-
tion of different approaches that can be adjusted to
fit the specific need of individual patients.
References
1. Adams DF. Diagnosis and treatment of refractory perio-
dontitis. Curr Opin Dent 1992: 2: 33–38.
2. Akizuki T, Oda S, Komaki M, Tsuchioka H, Kawakatsu N,
Kikuchi A, Yamato MJ, Okano T, Ishikawa I. Application of
periodontal ligament cell sheet for periodontal regener-
ation: a pilot study in beagle dogs. J Periodontal Res 2005:
40: 245–251.
3. Alvarez-Perez MA, Narayanan S, Zeichner-David M,
Rodriguez Carmona B, Arzate H. Molecular cloning,
expression and immunolocalization of a novel human
cementum-derived protein (CP-23). Bone 2006: 38: 409–
419.
4. Amar S, Chung KM, Nam SH, Karatzas S, Myokai F, Van
Dyke TE. Markers of bone and cementum formation
accumulate in tissues regenerated in periodontal defects
treated with expanded polytetrafluoroethylene mem-
branes. J Periodontal Res 1997: 32: 148–158.
5. Anitua E. Plasma rich in growth factors: preliminary results
of use in the preparation of future sites for implants. Int J
Oral Maxillofac Implants 1999: 14: 529–535.
6. Anusaksathien O, Webb SA, Jin QM, Giannobile WV
Platelet-derived growth factor gene delivery stimulates
ex vivo gingival repair. Tissue Eng 2003: 9: 745–756.
7. Anusaksathien O, Jin Q, ZhaoM, SomermanMJ, Giannobile
WV. Effect of sustained gene delivery of platelet-derived
growth factor or its antagonist (PDGF-1308) on tissue-
engineered cementum. J Periodontol 2004: 75: 429–440.
8. Arweiler NB, Auschill TM, Donos N, Sculean A. Antibac-
terial effect of an enamel matrix protein derivative on in
vivo dental biofilm vitality. Clin Oral Invest 2002: 6: 205–
209.
9. Arzate H, Portilla-Robertson J, Aguilar-Mendoza ME.
Recombination of epithelial root sheath and dental papilla
cells in vitro. Arch Med Res 1996: 27: 573–577.
10. Atkinson ME. The development of the mouse molar
periodontium. J Periodontal Res 1972: 7: 255–260.
11. BarKana I, Narayanan AS, Grosskop A, Savion N, Pitaru S.
Cementum attachment protein enriches putative cemen-
toblastic populations on root surfaces in vitro. J Dent Res
2000: 79: 1482–1488.
12. Bartold PM, Narayanan AS, Page RC. Platelet-derived
growth factor reduces the inhibitory effects of lipopoly-
saccharide on gingival fibroblast proliferation. J Periodon-
tal Res 1992: 27: 499–505.
13. Beertsen W, McCulloch CAG, Sodek J. The periodontal
ligament: a unique, multifunctional connective tissues.
Periodontol 2000 1997: 13: 20–40.
14. Berkovitz BK. Periodontal ligament: structural and clinical
correlates. Dent Update 2004: 31: 46–50.
15. Birek C, Heersche JN, Jez D, Brunette DM. Secretion of
a bone resorbing factor by epithelial cells cultured
from porcine rests of Malassez. J Periodontal Res 1983: 18:
75–81.
16. Birkedal-HansenH, ButlerWT, Taylor RE. Proteins from the
periodontium. characterization of the insoluble collagens of
bovine cementum. Calcif Tiss Res 1977: 23: 39–44.
17. Boabaid F, Gibson CW, Kuehl MA, Berry JE, Snead ML,
Nociti FH Jr, Katchburian E, Somerman MJ. Leucine-rich
amelogenin peptide: a candidate signaling molecule dur-
ing cementogenesis. J Periodontol 2004: 75: 1126–1136.
209
Regeneration of periodontal tissues: cementogenesis revisited
18. Borrell LN, Papapanou PN. Analytical epidemiology of
periodontitis. J Clin Periodontol 2005: 32: 132–158.
19. Bosshardt DD. Are cementoblasts a subpopulation of oste-
oblasts or a unique phenotype? J Dent Res 2005: 84: 390–406.
20. Bosshardt DD, Nanci A. Immunodetection of enamel- and
cementum-related (bone) proteins at the enamel-free area
and cervical portion of the tooth in rat molars. J Bone
Miner Res 1997: 12: 367–379.
21. Bosshardt DD, Selvig KA. Dental cementum: the dynamic
tissue covering the root. Periodontol 2000 1997: 13: 41–
75.
22. Bosshardt DD, Sculean A, Windisch P, Pjetursson BE, Lang
NP. Effects of enamel matrix proteins on tissue formation
along the roots of human teeth. J Periodontal Res 2005: 40:
158–167.
23. Boyan LA, Bhargava G, Nishimura F, Orman R, Price R,
Terranova VP. Mitogenic and hemotactic responses of
human periodontal ligament cells to the different isoforms
of platelet-derived growth factor. J Dent Res 1994: 73: 1593–
1600.
24. Brice GL, Sampson WJ, Sims MR. An ultrastructural eval-
uation of the relationship between epithelial rests of
Malassez and orthodontic root resorption and repair in
man. Aust Orthod J 1991: 12: 90–94.
25. Bronckers ALJJ, Farach-Carson MC, Van Waveren E, Butler
WT. Immunolocalization of osteopontin, osteocalcin and
dentin sialoproteins during dental root formation and early
cementogenesis in the rat. J Bone Mineral Res 1994: 9: 833–
841.
26. Browne RM. The pathogenesis of odontogenic cysts: a
review. J Oral Pathol 1975: 4: 31–46.
27. Brunette DM. Mechanical stretching increases the number
of epithelial cells synthesizing DNA in culture. J Cell Sci
1984: 69: 35–45.
28. Bunyaratavej P, Wang HL. Collagen membranes: a review.
J Periodontol 2001: 72: 215–229.
29. Caffesse RG, Holden MJ, Kon S, Nasjleti CE. The effect of
citric acid and fibronectin application on healing
following surgical treatment of naturally occurring perio-
dontal disease in beagle dogs. J Clin Periodontol 1985: 12:
578–590.
30. Camelo M, Nevins ML, Schenk RK, Lynch SE, Nevins M.
Periodontal regeneration in human Class II furcations
using purified recombinant human platelet-derived
growth factor-BB (rhPDGF-BB) with bone allograft. Int J
Periodontics Restorative Dent 2003: 23: 213–225.
31. Caton JG, Quinones CR. Etiology of periodontal diseases.
Curr Opin Dent 1991: 1: 17–28.
32. Caton JG, DeFuria EL, Polson AM, Nyman S. Periodontal
regeneration via selective cell repopulation. J Periodontol
1987: 58: 546–552.
33. Cattaneo V, Rota C, Silvestri M, Piacentini C, Forlino A,
Gallanti A, Rasperini G, Cetta G. Effect of enamel matrix
derivative on human periodontal fibroblasts: proliferation,
morphology and root surface colonization. An in vitro
study. J Periodontal Res 2003: 38: 568–574.
34. Chai Y, Jiang X, Ito Y, Bringas P, Han J, Rowitch DH,
Soriano P, McMahon AP, Sucov HM. Fate of the mam-
malian cranial neural crest during tooth and mandibular
morphogenesis. Development 2000: 127: 1671–1679.
35. Chano L, Tenenbaum HC, Lekic PC, Sodek J, McCulloch
CA. Emdogain regulation of cellular differentiation in
wounded rat periodontium. J Periodontal Res 2003: 38:
164–174.
36. Cho MI, Lin WL, Genco RJ. Platelet-derived growth factors-
modulated guided tissue regenerative therapy. J Perio-
dontol 1995: 66: 522–530.
37. Cochran DL, Wozney JM. Biological mediators for perio-
dontal regeneration. Periodontol 2000 1999: 19: 40–58.
38. D’Errico JA, Macneil RL, Takata T, Berry J, Strayhorn C,
Somerman MJ. Expression of bone associated markers by
tooth root lining cells, in situ and in vitro.Bone1997:20: 117–
126.
39. D’Souza RN, Cavender A, Sood R, Tarnuzzer R, Dickinson
DP, Roberts A, Letterio J. Dental abnormalities in mice
lacking a functional transforming factor-beta 1 (TGF-b1)
gene indicate a role for TGF-b1 in biomineralization. Int J
Oral Biol 1998: 23: 119–131.
40. Dassule HR, Lewis P, Bei M, Maas R, McMahon AP. Sonic
hedgehog regulates growth and morphogenesis of the
tooth. Development 2000: 127: 4775–4785.
41. Davenport DR, Mailhot JM, Wataha JC, Billman MA,
Sharawy MM, Shrout MK. Effects of enamel matrix
protein application on the viability, proliferation, and
attachment of human periodontal ligament fibroblasts to
diseased root surfaces in vitro. J Clin Periodontol 2003:
30: 125–131.
42. Davideau JL, Sahlberg C, Blin C, Papagerakis P, Thesleff I,
Berdal A. Differential expression of the full-length and
secreted truncated forms of EGF receptor during formation
of dental tissues. Int J Dev Biol 1995: 39: 605–615.
43. De Obarrio JJ, Arauz-Dutari JI, Chamberlain TM, Croston
A. The use of autologous growth factors in periodontal
surgical therapy: platelet gel biotechnology – case reports.
Int J Periodontics Restorative Dent 2000: 20: 486–497.
44. Dennison DK, Vallone DR, Pinero GJ, Rittman B, Caffesse
RG. Differential effect of TGF-beta 1 and PDGF on prolif-
eration of periodontal ligament cells and gingival fibro-
blasts. J Periodontol 1994: 65: 641–648.
45. Diekwisch TG. The developmental biology of cementum.
Int J Dev Biol 2001: 45: 695–706.
46. Diekwisch TG. Pathways and fate of migratory cells during
late tooth organogenesis. Connect Tissue Res 2002: 43: 245–
56.
47. Esposito M, Coulthard P, Worthington HV. Enamel matrix
derivative (Emdogain) for periodontal tissue regeneration
in intrabony defects. Cochrane Database Syst Rev 2003:
CD003875.
48. Feng F, Hou LT. Treatment of osseous defects with fibro-
blast-coated hydroxylapatite particles. J Formos Med Assoc
1992: 91: 1068–1074.
49. Feng F, Liu CM, Hsu WC, Hou LT. Long-term effects of
gingival fibroblast-coated hydroxylapatite graft on perio-
dontal osseous defects. J Formos Med Assoc 1995: 94: 494–
498.
50. Fetner AE, Hartigan MS, Low SB. Periodontal repair using
PerioGlas in nonhuman primates: clinical and histologic
observations. Compendium. 1994: 15: 935–938.
51. Fong HK, Foster BL, Popowics TE, Somerman MJ. The
crowning achievement: getting to the root of the problem.
J Dent Educ 2005: 69: 555–570.
52. Fowler EB, Breault LG, Cuenin MF. Periodontal disease
and its association with systemic disease. Mil Med 2001:
166: 85–89.
210
Zeichner-David
53. Franke Stenport V, Johansson CB. Enamel matrix deriv-
ative and titanium implants. J Clin Periodontol 2003: 30:
359–363.
54. Froum SJ, Weinberg MA, Rosenberg E, Tarnow D. A com-
parative study utilizing open flap debridement with and
without enamel matrix derivative in the treatment of per-
iodontal intrabony defects: a 12-month re-entry study.
J Periodontol 2001: 72: 25–34.
55. Fujiwara N, Tabata MJ, Endoh M, Ishizeki K, Nawa T.
Insulin-like growth factor-I stimulates cell proliferation in
the outer layer of Hertwig’s epithelial root sheath and
elongation of the tooth root in mouse molars in vitro. Cell
Tissue Res 2005: 320: 69–75.
56. Fujiyama K, Yamashiro T, Fukunaga T, Balam TA, Zheng L,
Takano-Yamamoto T. Denervation resulting in dento-
alveolar ankylosis associated with decreased Malassez
epithelium. J Dent Res 2004: 83: 625–629.
57. Gao Z, Mackenzie IC, Rittman BR, Korszun AK, Williams
DM, Cruchley AT. Immunocytochemical examination of
immune cells in periapical granulomata and odontogenic
cysts. J Oral Pathol 1988: 17: 84–90.
58. Gao J, Symons AL, Bartold PM. Expression of transforming
growth factor-beta 1 (TGF-beta1) in the developing peri-
odontium of rats. J Dent Res 1998: 77: 1708–1716.
59. Gao J, Symons AL, Bartold PM. Expression of transforming
growth factor-beta receptors types II and III within various
cells in the rat periodontium. J Periodontal Res 1999: 34:
113–122.
60. Gestrelius S, Lyngstadaas SP, Hammarstrom L. Emdogain–
periodontal regeneration based on biomimicry. Clin Oral
Invest 2000: 4: 120–125.
61. Giannobile WV. Periodontal tissue engineering by growth
factors. Bone 1996: 19: 23S–37S.
62. Giannobile WV, Somerman MJ. Growth and amelogenin-
like factors in periodontal wound healing. A systematic
review. Ann Periodontol 2003: 8: 193–204.
63. Giannobile WV, Finkelman RD, Lynch SE. Comparison of
canine and nonhuman primate animal models for perio-
dontal therapy. Results following a single administration of
PDGF/IGF-I. J Periodontol 1994: 65: 1158–1168.
64. Giannobile WV, Hernandez RA, Finkelman RD, Ryan S,
Kiritsy CP, D’Andrea M, Lynch SE. Comparative effects of
platelet-derived growth factor-BB and insulin-like growth
factor-I, individually and in combination, on periodontal
regeneration in Macaca fascicularis. J Periodontal Res 1996:
31: 301–12.
65. Giannobile WV, Lee CS, Tomala MP, Tejeda KM, Zhu Z.
Platelet-derived growth factor (PDGF) gene delivery for
application in periodontal tissue engineering. J Periodontol
2001: 72: 815–823.
66. Gottlow J. Guided tissue regeneration using bioresorbable
and non-resorbable devices: initial healing and long-term
results. J Periodontol 1993: 64: 1157–1165.
67. Gottlow J, Nyman S, Karring T, Lindhe J. New attachment
formation as the result of controlled tissue regeneration.
J Clin Periodontol 1984: 11: 494–503.
68. Graves DT, Kang YM, Kose KN. Growth factors in perio-
dontal regeneration. Compend Suppl 1994: 18: S672–S677.
69. Graziani F, Laurell L, Tonetti M, Gottlow J, Berglundh T.
Periodontal wound healing following GTR therapy of de-
hiscence-type defects in the monkey: short-, medium- and
long-term healing. J Clin Periodontol 2005: 32: 905–914.
70. Greenstein G. Emdogain: evidence of efficacy. Compend
Contin Educ Dent 2000: 21: 299–305.
71. Grzesik WJ, Narayanan AS. Cementum and periodontal
wound healing and regeneration. Crit Rev Oral Biol Med
2002: 13: 474–484.
72. Gurpinar A, Onur MA, Cehreli ZC, Tasman F. Effect of
enamel matrix derivative on mouse fibroblasts and marrow
stromal osteoblasts. J Biomater Appl 2003: 18: 25–33.
73. Haase HR, Bartold PM. Enamel matrix derivative induces
matrix synthesis by cultured human periodontal fibroblast
cells. J Periodontol 2001: 72: 341–348.
74. Hakki SS, Berry JE, Somerman MJ. The effect of enamel
matrix protein derivative on follicle cells in vitro. J Peri-
odontol 2001: 72: 679–687.
75. Hamamoto Y, Nakajima T, Ozawa H. Ultrastructural and
histochemical study on the morphogenesis of epithelial
rests of Malassez. Arch Histol Cytol 1989: 52: 61–70.
76. Hamamoto Y, Nakajima T, Ozawa H, Uchida T. Production
of amelogenin by enamel epithelium of Hertwig’s root
sheath. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
1996: 81: 703–709.
77. Hamamoto Y, Hamamoto N, Nakajima T, Ozawa H. Mor-
phological changes of epithelial rests of Malassez in rat
molars induced by local administration of N-methy-
lnitrosourea. Arch Oral Biol 1998: 43: 899–906.
78. Hammarstrom L. Enamel matrix, cementum develop-
ment and regeneration. J Clin Periodontol 1997: 24: 658–
668.
79. Hammarstrom L, Heijl L, Gestrelius S. Periodontal regen-
eration in a buccal dehiscence model in monkeys after
application of enamel matrix proteins. J Clin Periodontol
1997: 24: 669–677.
80. Harrison JW, Roda RS. Intermediate cementum. Develop-
ment, structure, composition, and potential functions.Oral
Surg OralMedOral Pathol Oral Radiol Endod 1995: 79: 624–
633.
81. Hasegawa N, Kawaguchi H, Ogawa T, Uchida T, Kurihara
H. Immunohistochemical characteristics of epithelial cell
rests of Malassez during cementum repair. J Periodontal
Res 2003: 38: 51–56.
82. Hassell TM. Tissues and cells of the periodontium.
Periodontol 2000 1993: 3: 9–38.
83. Heard RH, Mellonig JT. Regenerative materials: an
overview. Alpha Omegan 2000: 93: 51–58.
84. Heden G, Wennstrom J, Lindhe J. Periodontal tissue
alterations following Emdogain treatment of periodontal
sites with angular bone defects. A series of case reports.
J Clin Periodontol 1999: 26: 855–860.
85. Heijl L. Periodontal regeneration with enamel matrix
derivative in one human experimental defect. A case
report. J Clin Periodontol 1997: 24: 693–696.
86. Helder MN, Karg H, Bervoets TJ, Vukicevic S, Burger EH,
D’Souza RN, Woltgens JH, Karsenty G, Bronckers AL. Bone
morphogenetic protein-7 (osteogenic protein-1, OP-1) and
tooth development. J Dent Res 1998: 77: 545–554.
87. Hirooka H. The biologic concept for the use of enamel
matrix protein: true periodontal regeneration. Quintessence
Int 1998: 10: 621–630.
88. Ho SP, Sulyanto RM, Marshall SJ, Marshall GW. The
cementum-dentin junction also contains glycosami-
noglycans and collagen fibrils. J Struct Biol 2005: 151:
69–78.
211
Regeneration of periodontal tissues: cementogenesis revisited
89. Hoang AM, Oates TW, Cochran DL. In vitro wound healing
responses to enamel matrix derivative. J Periodontol 2000:
71: 1270–1277.
90. Hoang AM, Klebe RJ, Steffensen B, Ryu OH, Simmer JP,
Cochran DL. Amelogenin is a cell adhesion protein. J Dent
Res 2002: 81: 497–500.
91. Hou LT, Tsai AY, Liu CM, Feng F. Autologous transplan-
tation of gingival fibroblast-like cells and a hydroxylapatite
complex graft in the treatment of periodontal osseous
defects: cell cultivation and long-term report of cases. Cell
Transplant 2003: 12: 787–797.
92. Howell TH, Fiorellini JP, Paquette DW, Offenbacher S,
Giannobile WV, Lynch SE. A phase I/II clinical trial to
evaluate a combination of recombinant human platelet-
derived growth factor-BB and recombinant human insulin-
like growth factor-I in patients with periodontal disease.
J Periodontol 1997: 68: 1186–1193.
93. Huang KK, Shen C, Chiang CY, Hsieh YD, Fu EJ. Effects of
bone morphogenetic protein-6 on periodontal wound
healing in a fenestration defect of rats. J Periodontal Res
2005: 40: 1–10.
94. Igarashi R, Sahara T, Shimizu-Ishiura M, Sasaki T. Porcine
enamel matrix derivative enhances the formation of
reparative dentine and dentine bridges during wound
healing of amputated rat molars. J Electron Microsc 2003:
52: 227–236.
95. Inoue M, LeGeros RZ, Hoffman C, Diamond K, Rosenberg
PA, Craig RG. Effect of enamel matrix proteins on the phe-
notype expression of periodontal ligament cells cultured on
dental materials. J Biomed Mater Res 2004: 69A: 172–179.
96. Ishizaki NT, Matsumoto K, Kimura Y, Wang X, Yamashita
A. Histopathological study of dental pulp tissue capped
with enamel matrix derivative. J Endod 2003: 29: 176–179.
97. Ivanovski S, Li H, Haase HR, Bartold PM. Expression of
bone associated macromolecules by gingival and perio-
dontal ligament fibroblasts. J Periodontal Res 2001: 36:
131–141.
98. Iwata T, Morotome Y, Tanabe T, Fukae M, Ishikawa I, Oida
S. Noggin blocks osteoinductive activity of porcine enamel
extracts. J Dent Res 2002: 81: 387–391.
99. Jepsen S, Eberhard J, Herrera D, Needleman I. A systematic
review of guided tissue regeneration for periodontal furca-
tion defects. What is the effect of guided tissue regeneration
compared with surgical debridement in the treatment of
furcation defects? J Clin Periodontol 2002: 29: 103–116.
100. Jernvall J, Thesleff I. Reiterative signaling and patterning
during mammalian tooth morphogenesis. Mech Dev 2000:
92: 19–29.
101. Jiang J, Safavi KE, Spangberg LS, Zhu Q. Enamel matrix
derivative prolongs primary osteoblast growth. J Endod
2001: 27: 110–112.
102. Jiang J, Fouad AF, Safavi KE, Spangberg LS, Zhu Q. Effects
of enamel matrix derivative on gene expression of primary
osteoblasts. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 2001: 91: 95–100.
103. Jin QM, Anusaksathien O, Webb SA, Rutherford RB, Gian-
nobile WV. Gene therapy of bone morphogenetic protein
for periodontal tissue engineering. J Periodontol 2003: 74:
202–213.
104. Jin Q, Anusaksathien O, Webb SA, Printz MA, Giannobile
WV. Engineering of tooth-supporting structures by delivery
of PDGF gene therapy vectors. Mol Ther 2004: 9: 519–526.
105. Kagayama M, Sasano Y, Zhu JX, Hirata M, Mizohuchi I,
Kamakura S. Epithelial rest colocalize with cementoblasts
forming acellular cementum but not with cementoblasts
forming cellular cementum. Acta Anat 1998: 163: 1–9.
106. Kalajzic I, Braut A, Guo D, Jiang X, Kronenberg MS, Mina
M, Harris MA, Harris SE, Rowe DW. Dentin matrix protein
1 expression during osteoblastic differentiation, generation
of an osteocyte GFP-transgene. Bone 2004: 35: 74–82.
107. Kalpidis CD, Ruben MP. Treatment of intrabony perio-
dontal defects with enamel matrix derivative: a literature
review. J Periodontol 2002: 73: 1360–1376.
108. Kaneko H, Hashimoto S, Enokiya Y, Ogiuchi H, Shimono
M. Cell proliferation and death of Hertwig’s epithelial root
sheath in the rat. Cell Tissue Res 1999: 298: 95–103.
109. Karatzas S, Zavras A, Greenspan D, Amar S. Histologic
observations of periodontal wound healing after treatment
with PerioGlas in nonhuman primates. Int J Periodontics
Restorative Dent 1999: 19: 489–499.
110. Karlan MS, Hench LL, Madden M, Ogino M. A bone-
bonding bioactive material implant in the head and neck:
bioglass. Surg Forum 1978: 29: 575–577.
111. Kawaguchi H, Hirachi A, Hasegawa N, Iwata T, Hamaguchi
H, Shiba H, Takata T, Kato Y, Kurihara H. Enhancement of
periodontal tissue regeneration by transplantation of bone
marrow mesenchymal stem cells. J Periodontol 2004: 75:
1281–1287.
112. Kawase T, Okuda K, Yoshie H, Burns DM. Cytostatic action
of enamel matrix derivative (EMDOGAIN) on human oral
squamous cell carcinoma-derived SCC25 epithelial cells.
J Periodontal Res 2000: 35: 291–300.
113. Kawase T, Okuda K, Momose M, Kato Y, Yoshie H, Burns
DM. Enamel matrix derivative (EMDOGAIN) rapidly sti-
mulates phosphorylation of the MAP kinase family and
nuclear accumulation of smad2 in both oral epithelial and
fibroblastic human cells. J Periodontal Res 2001: 36: 367–
376.
114. Kawase T, Okuda K, Yoshie H, Burns DM. Anti-TGF-beta
antibody blocks enamel matrix derivative-induced upreg-
ulation of p21WAF1/cip1 and prevents its inhibition of
human oral epithelial cell proliferation. J Periodontal Res
2002: 37: 255–262.
115. Keila S, Nemcovsky CE, Moses O, Artzi Z, Weinreb M. In
vitro Effects of Enamel Matrix Proteins on Rat Bone Mar-
row Cells and Gingival Fibroblasts. J Dent Res 2004: 83:
134–138.
116. King GN. New regenerative technologies: rationale and
potential for periodontal regeneration: 2. Growth factors.
Dent Update 2001: 28: 60–65.
117. King GN. New regenerative technologies: rationale and
potential for periodontal regeneration: 1. New advances in
established regenerative strategies. Dent Update 2001: 28:
7–12.
118. King GN, Cochran DL. Factors that modulate the effects of
bone morphogenetic protein-induced periodontal regen-
eration: a critical review. J Periodontol 2002: 73: 925–936.
119. King GN, Hughes FJ. Bone morphogenetic protein-2 sti-
mulates cell recruitment and cementogenesis during early
wound healing. J Clin Periodontol 2001: 28: 465–475.
120. King GN, King N, Cruchley AT, Wozney JM, Hughes FJ.
Recombinant human bone morphogenetic protein-2 pro-
motes wound healing in rat periodontal fenestration de-
fects. J Dent Res 1997: 76: 1460–1470.
212
Zeichner-David
121. King GN, King N, Hughes FJ. Effect of two delivery systems
for recombinant human bone morphogenetic protein-2 on
periodontal regeneration in vivo. J Periodontal Res 1998:
33: 226–236.
122. Kittel PW, Sampson WJ. RME-induced root resorption and
repair: a computerised 3-D reconstruction. Aust Orthod J
1994: 13: 144–151.
123. Kobayashi M, Takiguchi T, Suzuki R, Yamaguchi A, Degu-
chi K, Shionome M, Miyazawa Y, Nishihara T, Nagumo M,
Hasegawa K. Recombinant human bone morphogenetic
protein-2 stimulates osteoblastic differentiation in cells
isolated from human periodontal ligament. J Dent Res
1999: 78: 1624–1633.
124. Koike H, Uzawa K, Grzesik WJ, Seki N, Endo Y, Kasamatsu
A, Yamauchi M, Tanzawa H. GLUT1 is highly expressed in
cementoblasts but not in osteoblasts. Connect Tissue Res
2005: 46: 117–124.
125. Kostopoulos L, Karring T. Susceptibility of GTR-regener-
ated periodontal attachment to ligature-induced perio-
dontitis. J Clin Periodontol 2004: 31: 336–340.
126. Lambrichts I, Creemers J, Van Steenberghe D. Periodontal
neural endings intimately relate to epithelial rests of Mal-
assez in humans. A light and electron microscope study.
J Anat 1993: 182: 153–162.
127. Lamster IB. Current concepts and future trends for perio-
dontal disease and periodontal therapy, Part 2: Classifica-
tion, diagnosis, and nonsurgical and surgical therapy. Dent
Today 2001: 20: 86–91.
128. Lamster IB, Karabin SD. Periodontal disease activity. Curr
Opin Dent 1992;2: 39–52.
129. Lang H, Schuler N, Nolden R. Attachment formation fol-
lowing replantation of cultured cells into periodontal de-
fects – a study in minipigs. J Dent Res 1998: 77: 393–405.
130. Lekic PC, Rajshankar D, Chen H, Tenenbaum H, McCul-
loch CA. Transplantation of labeled periodontal ligament
cells promotes regeneration of alveolar bone. Anat Rec
2001: 262: 193–202.
131. Lekic PC, Nayak BN, Al-Sanea R, Tenenbaum H, Ganss B,
McCulloch C. Cell transplantation in wounded mixed
connective tissues. Anat Rec A Discov Mol Cell Evol Biol
2005: 287: 1256–1263.
132. Lezot F, Davideau JL, Thomas B, Sharpe P, Forest N, Berdal
A. Epithelial Dlx-2 homeogene expression and cemento-
genesis. J Histochem Cytochem 2000: 48: 277–284.
133. Lindskog S, Hammarstrom L. Formation of intermediate
cementum III. 3H-tryptophan and 3H-proline uptake into
the epithelial root sheath of Hertwig in vitro. J Craniofac
Genet Dev Biol 1982: 2: 171–177.
134. Lindskog S, Blomlof L, Hammarstrom L. Evidence for a role
of odontogenic epithelium in maintaining the periodontal
space. J Clin Periodontol 1988: 15: 371–373.
135. Listgarten MA. Similarity of epithelial relationships in the
gingiva of rat and man. J Periodontol 1975: 46: 677–680.
136. Lubbock MJ, Harrison VT, Lumsden AG, Palmer RM.
Development and cell fate in interspecific (Mus musculus/
Mus caroli) intraocular transplants of mouse molar tooth-
germ tissues detected by in situ hybridization. Arch Oral
Biol 1996: 41: 77–84.
137. Lukinmaa PL, Mackie EJ, Thesleff I. Immunohistochemical
localization of the matrix glycoprotein-tenascin and the
ED-sequence form of cellular fibronectin. J Dent Res 1991:
70: 19–26.
138. Lynch SE, Ruiz de Castilla G, Williams RC, Kiritsy CP,
Howell TH, Reddy MS, Antoniades HN. The effects of
short-term application of a combination of platelet-de-
rived and insulin-like growth factors on periodontal wound
healing. J Periodontol 1991: 62: 458–467.
139. Lyngstadaas SP, Lundberg E, Ekdahl H, Andersson C,
Gestrelius S. Autocrine growth factors in human perio-
dontal ligament cells cultured on enamel matrix derivative.
J Clin Periodontol 2001: 28: 181–188.
140. MacNeil RL, Thomas HF. Development of the murine
periodontium. I. role of basement membrane in formation
of a mineralized tissue on the developing root dentin
surface. J Periodontol 1993: 64: 95–102.
141. MacNeil RL, Thomas HF. Development of the murine
periodontium. II. Role of the epithelial root sheath in for-
mation of the periodontal attachment. J Periodontol 1993:
64: 285–291.
142. Macneil RL, Berry J, Strayhorn C, Somerman MJ. Expres-
sion of bone sialoprotein mRNA by cells lining the mouse
tooth root during cementogenesis. Arch Oral Biol 1996: 41:
827–835.
143. Madan AK, Kramer B. Immunolocalization of fibroblast
growth factor-2 (FGF-2) in the developing root and sup-
porting structures of the murine tooth. J Mol Histol 2005:
36: 171–178.
144. Mariotti A. The extracellular matrix of the periodontium:
dynamic and interactive tissues. Periodontol 2000 1993: 3:
39–63.
145. Mariotti A Efficacy of chemical root surface modifiers in
the treatment of periodontal disease. A systematic review.
Ann Periodontol 2003: 8: 205–226.
146. Matsuda N, Lin WL, Kumar NM, Cho MI, Genco RJ.
Mitogenic, chemotactic, and synthetic responses of rat
periodontal ligament fibroblastic cells to polypeptide
growth factors in vitro. J Periodontol 1992: 63: 515–525.
147. Matsuura M, Herr Y, Han KY, Lin WL, Genco RJ, Cho MI.
Immunohistochemical expression of extracellular matrix
components of normal and healing periodontal tissues in
the beagle dog. J Periodontol 1995: 66: 579–593.
148. Maycock J, Wood SR, Brookes SJ, Shore RC, Robinson C,
Kirkham J. Characterization of a porcine amelogenin pre-
paration, EMDOGAIN, a biological treatment for perio-
dontal disease. Connect Tissue Res 2002: 43: 472–476.
149. McAllister B, Narayanan AS, Miki Y, Page RC. Isolation of a
fibroblast attachment protein from cementum. J Perio-
dontal Res 1990: 25: 99–105.
150. McCulloch CA Origins and functions of cells essential for
periodontal repair: the role of fibroblasts in tissue home-
ostasis. Oral Dis 1995: 1: 271–278.
151. McCulloch CA, Lekic P, McKee MD. Role of physical forces
in regulating the form and function of the periodontal
ligament. Periodontol 2000 2000: 24: 56–72.
152. Melcher AH On the repair potential of periodontal tissues.
J Periodontol 1976: 47: 256–260.
153. Melcher AH. Cells of periodontium: their role in the heal-
ing of wounds. Ann R Coll Surg Engl 1985: 67: 130–131.
154. Mellonig JT Enamel matrix derivative for periodontal re-
constructive surgery: technique and clinical and histologic
case report. Int J Periodontics Restorative Dent 1999: 19:
8–19.
155. Merrilees MJ, Sodek J, Aubin JE. Effects of cells of epithelial
rests of Malassez and endothelial cells on synthesis of
213
Regeneration of periodontal tissues: cementogenesis revisited
glycosaminoglycans by periodontal ligament fibroblasts in
vitro. Dev Biol 1983: 97: 146–153.
156. Metzger Z, Weinstock B, Dotan M, Narayanan AS, Pitaru S.
Differential chemotactic effect of cementum attachment
protein on periodontal cells. J Periodontal Res 1998: 33:
126–129.
157. Miki Y, Narayanan AS, Page RC. Mitogenic activity of
cementum components to gingival fibroblasts. J Dent Res
1987: 66: 1399–1403.
158. Miletich I, Sharpe PT. Neural crest contribution to mam-
malian tooth formation. Birth Defects Res C Embryo Today
2004: 72: 200–212.
159. Miller N, Penaud J, Foliguet B, Membre H, Ambrosini P,
Plombas M. Resorption rates of 2 commercially available
bioresorbable membranes. A histomorphometric study in
a rabbit model. J Clin Periodontol 1996: 23: 1051–1059.
160. Mirastschijski U, Konrad D, Lundberg E, Lyngstadaas SP,
Jorgensen LN, Agren MS. Effects of a topical enamel matrix
derivative on skin wound healing. Wound Repair Regen
2004: 12: 100–108.
161. Mizutani S, Tsuboi T, Tazoe M, Koshihara Y, Goto S, Togari
A. Involvement of FGF-2 in the action of Emdogain on
normal human osteoblastic activity. Oral Dis 2003: 9: 210–
217.
162. Morsczeck C, Gotz W, Schierholz J, Zeilhofer F, Kuhn U,
Mohl C, Sippel C, Hoffmann KH. Isolation of precursor
cells (PCs) from human dental follicle of wisdom teeth.
Matrix Biol 2005: 24: 155–165.
163. Murphy KG, Gunsolley JC. Guided tissue regeneration for
the treatment of periodontal intrabony and furcation
defects. A systematic review. Ann Periodontol 2003: 8: 266–
302.
164. Murphy WL, Mooney DJ. Controlled delivery of inductive
proteins, plasmid DNA and cells from tissue engineering
matrices. J Periodontal Res 1999: 34: 413–419.
165. Nabers CL, Pfeifer JS, Raust GT Jr, Ross SJ. What is the
place of bone grafts in periodontal therapy? Periodontal
Abstr 1967: 5: 149–153.
166. Nagano T, Iwata T, Ogata Y, Tanabe T, Gomi K, Fukae M,
Arai T, Oida S. Effect of heat treatment on bioactivities of
enamel matrix derivatives in human periodontal liga-
ment(HPDL) cells. J Periodontal Res 2004: 39: 249–256.
167. Nakae H, Narayanan S, Raines E, Page RC. Isolation and
partial characterization of mitogenic factors from cemen-
tum. Biochemistry 1991: 30: 7047–7052.
168. Nakamura Y, Hammarstrom L, Lundberg E, Ekdahl H,
Matsumoto K, Gestrelius S, Lyngstadaas SP. Enamel matrix
derivative promotes reparative processes in the dental
pulp. Adv Dent Res 2001: 15: 105–107.
169. Nakamura Y, Hammarstrom L, Matsumoto K, Lyngstadaas
SP. The induction of reparative dentine by enamel pro-
teins. Int Endod J 2002: 35: 407–417.
170. Nakashima M, Reddi AH. The application of bone mor-
phogenetic proteins to dental tissue engineering. Nat
Biotechnol 2003: 21: 1025–1032.
171. Nevins M, Camelo M, Nevins ML, Schenk RK, Lynch SE.
Periodontal regeneration in humans using recombinant
human platelet-derived growth factor-BB (rhPDGF-BB)
and allogenic bone. J Periodontol 2003: 74: 1282–1292.
172. Newman SA, Coscia SA, Jotwani R, Iacono VJ, Cutler CW.
Effects of enamel matrix derivative on Porphyromonas
gingivalis. J Periodontol 2003: 74: 1191–1195.
173. Nishimura K, Hayahi M, Matsuda K, Shigeyama Y,
Yamasaki A, Yamaoka A. The chemoattractive potency
of periodontal ligament, cementum and dentin for
human gingival fibroblasts. J Periodontal Res 1989: 24:
146–148.
174. Nyman S, Gottlow J, Karring T, Lindhe J. The regenerative
potential of the periodontal ligament. An experimental
study in the monkey. J Clin Periodontol 1982: 9: 257–265.
175. Oates TW, Rouse CA, Cochran DL. Mitogenic effects of
growth factors on human periodontal ligament cells in
vitro. J Periodontol 1993: 64: 142–148.
176. Ohshima M, Nishiyama T, Tokunaga K, Sato S, Maeno M,
Otsuka K. Profiles of cytokine expression in radicular cyst-
lining epithelium examined by RT-PCR. J Oral Sci 2000: 42:
239–246.
177. Ohyama M, Suzuki N, Yamaguchi Y, Maeno M, Otsuka K,
Ito K. Effect of enamel matrix derivative on the differenti-
ation of C2C12 cells. J Periodontol 2002: 73: 543–550.
178. Okubo K, Kobayashi M, Takiguchi T, Takada T, Ohazama
A, Okamatsu Y, Hasegawa K. Participation of endogenous
IGF-I and TGF-beta 1 with enamel matrix derivative-sti-
mulated cell growth in human periodontal ligament cells.
J Periodontal Res 2003: 38: 1–9.
179. Okuda K, Momose M, Miyazaki A, Murata M, Yokoyama S,
Yonezawa Y, Wolff LF, Yoshie H. Enamel matrix derivative
in the treatment of human intrabony osseous defects.
J Periodontol 2000: 71: 1821–1828.
180. Okuda K, Kawase T, Momose M, Murata M, Saito Y, Suzuki
H, Wolff LF, Yoshie H. Platelet-rich plasma contains high
levels of platelet-derived growth factor and transforming
growth factor-beta and modulates the proliferation of
periodontally related cells in vitro. J Periodontol 2003: 74:
849–857.
181. Orban B. The epithelial network in the periodontal mem-
brane. J Am Dent Assoc 1952: 44: 632–635.
182. Owens PDA. Ultrastructure of Hertwig’s epithelial root
sheath during early root development in premolar teeth in
dogs. Arch Oral Biol 1978: 23: 91–104.
183. Owens PDA. A light and electron microscopic study of
early stages of root surface formation in molar teeth in the
rat. Arch oral Biol 1980: 24: 901–907.
184. Palioto DB, Coletta RD, Graner E, Joly JC, de Lima AF. The
influence of enamel matrix derivative associated with
insulin-like growth factor-I on periodontal ligament fi-
broblasts. J Periodontol 2004: 75: 498–504.
185. Palmer RM, Lubbock MJ. The soft connective tissues of the
gingiva and periodontal ligament: are they unique? Oral
Dis 1995: 1: 230–237.
186. Palmer RM, Lumsden AG. Development of periodontal
ligament and alveolar bone in homografted recombina-
tion’s of enamel organs and papillary, pulpal, and follicular
mesenchyme in the mouse. Arch Oral Biol 1987: 32: 281–
289.
187. Parashis A, Tsiklakis K. Clinical and radiographic findings
following application of enamel matrix derivative in the
treatment of intrabony defects. A series of case reports.
J Clin Periodontol 2000: 27: 705–713.
188. Park JB, Matsuura M, Han KY, Norderyd O, Lin WL, Genco
RJ. Periodontal regeneration in class III furcation defects in
beagle dogs using guided tissue regenerative therapy with
platelet-derived growth factor. J Periodontol 1995: 66: 462–
477.
214
Zeichner-David
189. Parodi R, Liuzzo G, Patrucco P, Brunel G, Santarelli GA,
Birardi V, Gaspretto B. Use of Emdogain in the treatment
of deep intrabony defects: 12-month clinical results. His-
tologic and radiographic evaluation. Int J Periodontics
Restorative Dent 2000: 6: 584–595.
190. Parodi R, Santarelli GA, Gasparetto B. Treatment of intra-
bony pockets with Emdogain: results at 36 months. Int J
Periodontics Restorative Dent 2004: 24: 57–63.
191. Paynter KJ, Pudy G. A study of the structure, chemical
nature, and development of cementum in the rat. Anat Rec
1958: 131: 233–251.
192. Peters H, Balling R. Teeth, where and how to make them.
Trends Genet 1999: 15: 59–64.
193. Phipps RP, Borrello MA, Blieden TMJ. Fibroblast hetero-
geneity in the periodontium and other tissues. J Perio-
dontal Res 1997: 32: 159–165.
194. Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal
diseases. Lancet 2005: 366: 1809–1820.
195. Pitaru S, McCulloch CA, Narayanan AS. Cellular origins
and differentiation control mechanisms during periodon-
tal development and wound healing. J Periodontal Res
1994: 29: 81–94.
196. Pitaru S, Narayanan SA, Olsen S, Avion N, Hekmati H, Alt I,
Metzger Z. Specific cementum attachment protein
enhances selectively the attachment and migration of
periodontal cells to root surfaces. J Periodontal Res 1995:
30: 360–368.
197. Plemons JM, Dill RE, Rees TD, Dyer BJ, Ng MC, Iacopino
AM. PDGF-B producing cells and PDGF-B gene expression
in normal gingival and cyclosporine A-induced gingival
overgrowth. J Periodontol 1996: 67: 264–270.
198. Plikus MV, Zeichner-David M, Mayer JA, Reyna J, Bringas
P, Thewissen JG, Snead ML, Chai Y, Chuong CM. Mor-
phoregulation of teeth: modulating the number, size,
shape and differentiation by tuning Bmp activity. Evol Dev
2005: 7: 440–457.
199. Price JA, Wright JT, Kula K, Bowden DW, Hart TC. A
common DLX3 gene mutation is responsible for tricho-
dento-osseous syndrome in Virginia and North Carolina
families. J Med Genet 1998: 35: 825–828.
200. Rasperini G, Ricci G, Silvestri M. Surgical technique for
treatment of infrabony defects with enamel matrix deriv-
ative (Emdogain): 3 case reports. Int J Periodontics
Restorative Dent 1999: 19: 578–587.
201. Reichert T, Storkel S, Becker K, Fisher LW. The role of
osteonectin in human tooth development: an immuno-
histological study. Calc Tissue Int 1992: 50: 468–472.
202. Reynolds MA, Aichelmann-Reidy ME, Branch-Mays GL,
Gunsolley JC. The efficacy of bone replacement grafts in
the treatment of periodontal osseous defects. A systematic
review. Ann Periodontol 2003: 8: 227–265.
203. Rincon JC, Haase HR, Bartold PM. Effect of Emdogain on
human periodontal fibroblasts in an in vitro wound-heal-
ing model. J Periodontal Res 2003: 38: 290–295.
204. Rincon JC, Xiao Y, Young WG, Bartold PM. Enhanced
proliferation, attachment and osteopontin expression by
porcine periodontal cells exposed to Emdogain. Arch Oral
Biol 2005: 50: 1047–1054.
205. Ripamonti U, Reddi AH. Tissue engineering, morphogen-
esis, and regeneration of the periodontal tissues by bone
morphogenetic proteins. Crit Rev Oral Biol Med 1997: 8:
154–163.
206. Ripamonti U, Petit JC, Lemmer J, Austin JC. Regeneration
of the connective tissue attachment on surgically exposed
roots using fibrin-fibronectin adhesive system. An experi-
mental study on the baboon (Papio ursinus). J Periodontal
Res 1987: 22: 320–326.
207. Ripamonti U, Crooks J, Petit JC, Rueger DC. Periodontal
tissue regeneration by combined applications of recom-
binant human osteogenic protein-1 and bone morpho-
genetic protein-2. A pilot study in Chacma baboons (Papio
ursinus). Eur J Oral Sci 2001: 109: 241–248.
208. Robey PG, Bianco P, Termine JD. The cellular biology and
molecular biochemistry of bone formation. In: Coe FI,
Favuus MJ, editors. Disorders of Bone and Mineral Meta-
bolism. New York: Raven Press, 1992: 241–263.
209. Rose LF, Rosenberg E. Bone grafts and growth and differ-
entiation factors for regenerative therapy: a review. Pract
Proced Aesthet Dent 2001: 13: 725–734.
210. Rutherford RB, Niekrash CE, Kennedy JE, Charette MF.
Platelet-derived and insulin-like growth factors stimu-
late regeneration of periodontal attachment in monkeys.
J Periodontal Res 1992: 27: 285–290.
211. Rutherford RB, Ryan ME, Kennedy JE, Tucker MM,
Charette MF. Platelet-derived growth factor and dexa-
methasone combined with collagen matrix induce regen-
eration of the periodontium in monkeys. J Clin Periodontol
1993: 20: 537–544.
212. Saffar JL, Lasfargues JJ, Cherruau M. Alveolar bone and the
alveolar process: the socket that is never stable. Period-
ontol 2000 1997: 13: 76–90.
213. Sato Y, Kikuchi M, Ohata N, Tamura M, Kuboki Y. En-
hanced cementum formation in experimentally induced
cementum defects of the root surface with the application
of recombinant basic fibroblast growth factor in collagen
gel in vivo. J Periodontol 2004: 75: 243–248.
214. Schroeder HE, Listgarten MA. The gingival tissues: the
architecture of periodontal protection. Periodontol 2000
1997: 13: 91–120.
215. Schwartz Z, Carnes DL Jr, Pulliam R, Lohmann CH, Sylvia
VL, Liu Y, Dean DD, Cochran DL, Boyan BD. Porcine fetal
enamel matrix derivative stimulates proliferation but not
differentiation of pre-osteoblastic 2T9 cells, inhibits pro-
liferation and stimulates differentiation of osteoblast-like
MG63 cells, and increases proliferation and differentiation
of normal human osteoblast NHOst cells. J Periodontol
2000: 71: 1287–1296.
216. Sculean A, Donos N, Blaes A, Lauermann M, Reich E, Brecx
M. Comparison of enamel matrix proteins and bio-
absorbable membranes in the treatment of intrabony
periodontal defects. A split-mouth study. J Periodontol
1999: 70: 255–262.
217. Sculean A, Chiantella GC, Windisch P, Donos N. Clinical
and histologic evaluation of human intrabony defects
treated with an enamel matrix protein derivative (Emdo-
gain). Int J Periodontics Restorative Dent 2000: 20: 374–381.
218. Sculean A, Donos N, Brecx M, Reich E, Karring T. Treat-
ment of intrabony defects with guided tissue regeneration
and enamel-matrix-proteins. An experimental study in
monkeys. J Clin Periodontol 2000: 27: 466–472.
219. Sculean A, Donos N, Brecx M, Karring T, Reich E. Healing of
fenestration-type defects following treatment with guided
tissue regeneration or enamel matrix proteins. An experi-
mental study in monkeys. Clin Oral Investig 2000: 4: 50–56.
215
Regeneration of periodontal tissues: cementogenesis revisited
220. Sculean A, Auschill TM, Donos N, Brecx M, Arweiler NB.
Effect of an enamel matrix protein derivative (Emdogain)
on ex vivo dental plaque vitality. J Clin Periodontol 2001:
28: 1074–1078.
221. Sculean A, Windisch P, Keglevich T, Gera I. Clinical and
histologic evaluation of an enamel matrix protein deriv-
ative combined with a bioactive glass for the treatment of
intrabony periodontal defects in humans. Int J Periodontics
Restorative Dent 2005: 25: 139–147.
222. Selvig KA. Electron microscopy of Hertwig’s epithelial
sheath and of early dentin and cementum formation in the
mouse incisor. Acta Odontol Scand 1963: 21: 175–186.
223. Sena K, Morotome Y, Baba O, Terashima T, Takano Y,
Ishikawa I. Gene expression of growth differentiation fac-
tors in the developing periodontium of rat molars. J Dent
Res 2003: 82: 166–171.
224. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S,
Brahim J, Young M, Robey PG, Wang CY, Shi S. Investiga-
tion of multipotent postnatal stem cells from human per-
iodontal ligament. Lancet 2004: 364: 149–155.
225. Seo BM, Miura M, Sonoyama W, Coppe C, Stanyon R, Shi
S. Recovery of stem cells from cryopreserved periodontal
ligament. J Dent Res 2005: 84: 907–912.
226. Shear M, Pindborg JJ. Microscopic features of the lateral
periodontal cyst. Scand J Dent Res 1975: 83: 103–110.
227. Shimizu E, Nakajima Y, Kato N, Nakayama Y, Saito R,
Samoto H, Ogata Y. Regulation of rat bone sialoprotein
gene transcription by enamel matrix derivative. J Period-
ontol 2004: 75: 260–267.
228. Shimizu E, Saito R, Nakayama Y, Nakajima Y, Kato N, Takai
H, Kim DS, Arai M, Simmer J, Ogata Y. Amelogenin stimu-
lates bone sialoprotein (BSP) expression through fibroblast
growth factor 2 response element and transforming growth
factor-beta1 activation element in the promoter of the BSP
gene. J Periodontol 2005: 76: 1482–1489.
229. Shimizu-Ishiura M, Tanaka S, Lee WS, Debari K, Sasaki
T. Effects of enamel matrix derivative to titanium
implantation in rat femurs. J Biomed Mater Res 2002: 60:
269–276.
230. Shroff B, Kashner JE, Keyser JD, Hebert C, Norris K Epi-
dermal growth factor and epidermal growth factor-recep-
tor expression in the mouse dental follicle during tooth
eruption. Arch Oral Biol 1996: 41: 613–617.
231. Silvestri M, Rasperini G, Euwe E. Enamel matrix derivative
in treatment of infrabony defects. Pract Periodontics
Aesthet Dent 1999: 11: 615–616.
232. Slavkin HC. Towards a cellular and molecular under-
standing of periodontics: cementogenesis revisited. J Pe-
riodontol 1976: 47: 249–255.
233. Slavkin HC, Boyd A. Cementum: an epithelial secretory
product? J Dent Res 1974: 53: 157.
234. Slavkin HC, Bringas P, Bessem C, Santos V, Nakamura M,
Hsu M, Snead ML, Zeichner-David M, Fincham AG. Her-
twig’s epithelial root sheath differentiation and initial ce-
mentum and bone formation during long-term organ
culture of mouse mandibular first molars using serumless,
chemically-defined medium. J Periodontal Res 1988: 23:
28–40.
235. Slavkin HC, Bessem C, Fincham AG, Bringas P, Santos V,
Snead ML, Zeichner-David M. Human and mouse ce-
mentum proteins immunologically related to enamel
proteins. Biochim Biophys Acta 1989: 991: 12–18.
236. Somerman MJ, Perez-Mera M, Merkhofer RM, Foster RA.
In vitro evaluation of extracts of mineralized tissues for
their application in attachment of fibrous tissue. J Period-
ontol 1987: 58: 349–351.
237. Spahr A, Lyngstadaas SP, Boeckh C, Andersson C, Podb-
ielski A, Haller B. Effect of the enamel matrix derivative
Emdogain on the growth of periodontal pathogens in vitro.
J Clin Periodontol 2002: 29: 62–72.
238. Spouge JD. A new look at the rests of Malassez: a review of
their embryological origin, anatomy, and possible role in
periodontal health and disease. J Periodontol 1980: 51:
437–444.
239. Steele-Perkins G, Butz KG, Lyons GE, Zeichner-David M,
Kim HJ, Cho MI, Gronostajski RM. Essential role for NFI-C/
CTF transcription-replication factor in tooth root devel-
opment. Mol Cell Biol 2003: 23: 1075–1084.
240. Sykaras N, Opperman LA. Bone morphogenetic proteins
(BMPs): how do they function and what can they offer the
clinician? J Oral Sci 2003: 45: 57–73.
241. Talic NF, Evans CA, Daniel JC, Zaki AE. Proliferation of
epithelial rests of Malassez during experimental tooth
movement. Am J Orthod Dentofacial Orthop 2003: 123:
527–533.
242. Tatemoto Y, Okada Y, Mori M. Squamous odontogenic
tumor: immunohistochemical identification of keratins.
Oral Surg Oral Med Oral Pathol 1989: 67: 63–67.
243. Ten Cate AR. A fine structural study of coronal and root
dentinogenesis in the mouse. Observations of the so called
��von korff fibers�� and their contribution to mantle dentine.
J Anat 1978: 125: 183–197.
244. Ten Cate AR. The role of epithelium in the development,
structure and function of the tissues of tooth support. Oral
Dis 1996: 2: 55–62.
245. TenCateAR,Mills C. The development of the periodontium:
the origin of alveolar bone. Anat Rec 1972: 173: 69–78.
246. Tenorio D, Cruchley A, Hughes FJ. Immunocytochemical
investigation of the rat cementoblast phenotype. J Perio-
dontal Res 1993: 28: 411–419.
247. Terranova VP. Periodontal and bone regeneration factor,
materials and methods. International patent # WO 90/
100017. 1990.
248. Terranova VP, Wikesjo UM. Extracellular matrices and
polypeptide growth factors as mediators of functions of
cells of the periodontium. J Periodontol 1987: 58: 371–380.
249. Thesleff I. Developmental biology and building a tooth.
Quintessence Int 2003: 34: 613–620.
250. Thomadakis G, Ramoshebi LN, Crooks J, Rueger DC,
Ripamonti U. Immunolocalization of Bone Morphogenetic
Protein-2 and -3 and Osteogenic Protein-1 during murine
tooth root morphogenesis and in other craniofacial struc-
tures. Eur J Oral Sci 1999: 107: 368–377.
251. ThomasHF.Root formation. Int JDevBiol1995:39: 231–237.
252. Thomas HF, Kollar EJ. Differentiation of odontoblasts in
grafted recombinants of murine epithelial root sheath and
dental mesenchyme. Arch Oral Biol 1989: 34: 27–35.
253. Thomas BL, Tucker AS, Qui M, Ferguson CA, Hardcastle Z,
Rubenstein JL, Sharpe P. Role of Dlx-1 and Dlx-2 genes in
patterning of the murine dentition. Development 1997:
124: 4811–4818.
254. Tokiyasu Y, Takata T, Saygin E, Somerman M. Enamel
factors regulate expression of genes associated with ce-
mentoblasts. J Periodontol 2000: 71: 1829–1839.
216
Zeichner-David
255. Toyosawa S, Okabayashi K, Komori T, Ijuhin N. mRNA
expression and protein localization of dentin matrix pro-
tein 1 during dental root formation. Bone. 2004: 34: 124–
133.
256. Van der Pauw MT, Van den Bos T, Everts V, Beertsen W.
Enamel matrix-derived protein stimulates attachment of
periodontal ligament fibroblasts and enhances alkaline
phosphatase activity and transforming growth factor beta1
release of periodontal ligament and gingival fibroblasts.
J Periodontol 2000: 71: 31–43.
257. Venezia E, Goldstein M, Boyan BD, Schwartz Z. The use of
enamel matrix derivative in the treatment of periodontal
defects: a literature review and meta-analysis. Crit Rev Oral
Biol Med 2004: 15: 382–402.
258. Viswanathan HL, Berry JE, Foster BL, Gibson CW, Li Y,
Kulkarni AB, Snead ML, Somerman MJ. Amelogenin: a
potential regulator of cementum-associated genes. J Peri-
odontol 2003: 74: 1423–1431.
259. Wallace JA, Vergona K. Epithelial rests� function in re-
plantation: is splinting necessary in replantation? Oral Surg
Oral Med Oral Pathol 1990: 70: 644–649.
260. Wang HL, Greenwell H, Fiorellini J, Giannobile W, Offen-
bacher S, Salkin L, Townsend C, Sheridan P, Genco RJ.
Research, Science and Therapy Committee. Periodontal
regeneration. J Periodontol 2005: 76: 1601–1622.
261. Wesselink PR, Beertsen W. The prevalence and distribution
of Malassez in the mouse molar and their possible role in
repair and maintenance of the periodontal ligament. Arch
Oral Biol 1993: 88: 399–403.
262. Wikesjo UME, Claffey N, Christersson LA, Franzetti LC,
Genco RJ, Terranova VP. Repair of periodontal furcation
defects in beagle dogs following reconstructive surgery
including root surface de-mineralization with tetracycline
hydrochloride and topical fibronectin application. J Clin
Periodontol 1988: 15: 73–80.
263. Wu D-Y, Ikezawa K, Parker T, Saito M, Narayanan A-S.
Characterization of a collagenous cementum-derived
attachment. J Bone Mineral Res 1996: 11: 686–692.
264. Yamamoto H, Cho SW, Kim EJ, Kim JY, Fujiwara N, Jung
HS. Developmental properties of the Hertwig’s epithelial
root sheath in mice. J Dent Res 2004: 83: 688–692.
265. Yamamoto T, Domon T, Takahashi S, Arambawatta AK,
Wakita M. Immunolocation of proteoglycans and bone-
related noncollagenous glycoproteins in developing acel-
lular cementum of rat molars. Cell Tissue Res 2004: 317:
299–312.
266. Yamashiro T, Tummers M, Thesleff I. Expression of bone
morphogenetic proteins and Msx genes during root for-
mation. J Dent Res 2003: 82: 172–176.
267. Yamaza T, Kido MA, Tanaka T, Hashimoto S, Shimono M,
Ishikawa T, Enokiya Y, Muramatsu T, Matsuzaka K, Inoue
T, Abiko Y. Biological characteristics of the junctional
epithelium. J Electron Microsc 2003: 52: 627–639.
268. Yoneda S, Itoh D, Kuroda S, Kondo H, Umezawa A, Ohya K,
Ohyama T, Kasugai S. The effects of enamel matrix deriv-
ative (EMD) on osteoblastic cells in culture and bone
regeneration in a rat skull defect. J Periodontal Res 2003:
38: 333–342.
269. Yonemura K, Narayanan AS, Miki Y, Page RC, Okada H.
Isolation and partial characterization of a growth factor
from cementum. Bone Mineral Res 1992: 18: 187–198.
270. Young WG. Growth hormone and insulin-like growth fac-
tor-I in odontogenesis. Int J Dev Biol 1995: 39: 263–272.
271. Yuan K, Chen CL, Lin MT. Enamel matrix derivative
exhibits angiogenic effect in vitro and in a murine model.
J Clin Periodontol 2003: 30: 732–738.
272. Zeichner-David M. Is there more to enamel matrix proteins
than biomineralization? Matrix Biol 2001: 20: 307–316.
273. Zeichner-David M, Su Z, Chen L, Zakartchenko V, Caton J,
Bringas P. Effect of recombinant mouse amelogenin and
ameloblastin on periodontal ligament cell adhesion and
proliferation. EJOS (In press).
274. Zeichner-David M, Diekwisch T, Fincham A, Lau E, Mac-
Dougall M, Moradian-Oldak J, Simmer J, Snead M, Slavkin
HC. Control of ameloblast cell differentiation. Int J Develop
Biol 1995: 39: 69–92.
275. Zeichner-David M, Oishi K, Su Z, Zakartchenko V, Chen LS,
Arzate H, Bringas P Jr. Role of Hertwig’s epithelial root
sheath cells in tooth root development. Dev Dyn 2003: 228:
651–663.
276. Zernik JH, Nowroozi N, Liu YH, Maxson R. Development,
maturation, and aging of the alveolar bone. New insights.
Dent Clin North Am 1997: 41: 1–15.
277. Zetterstrom O, Andersson C, Eriksson L, Fredriksson A,
Friskopp J, Heden G, Jansson B, Lundgren T, Nilveus R,
Olsson A, Renvert S, Salonen L, Sjostrom L, Winell A,
Ostgren A, Gestrelius S. Clinical safety of enamel matrix
derivative (EMDOGAIN) in the treatment of periodontal
defects. J Clin Periodontol 1997: 24: 697–704.
278. Zhao M, Xiao G, Berry JE, Franceschi RT, Reddi A,
Somerman MJ. Bone morphogenetic protein 2 induces
dental follicle cells to differentiate toward a cementoblast/
osteoblast phenotype. J Bone Miner Res 2002: 17: 1441–
1451.
279. Zhao M, Berry JE, Somerman MJ. Bone morphogenetic
protein-2 inhibits differentiation and mineralization of
cementoblasts in vitro. J Dent Res 2003: 82: 23–27.
280. Zhao M, Jin Q, Berry JE, Nociti FH Jr, Giannobile WV,
Somerman MJ. Cementoblast delivery for periodontal tis-
sue engineering. J Periodontol 2004: 75: 154–161.
217
Regeneration of periodontal tissues: cementogenesis revisited