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

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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.

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