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Cell-to-cellcommunication–periodontalregeneration

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DieterBosshardt

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BerndStadlinger

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HendrikTerheyden

RedCrossHospital,Kassel,Germany

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Dieter D. BosshardtBernd StadlingerHendrik Terheyden

Cell-to-cell communication –periodontal regeneration

Authors’ affiliations:Dieter D. Bosshardt, Robert K. Schenk Laboratoryof Oral Histology, University of Bern, Bern,Switzerland, Department of Periodontology,University of Bern, Bern, Switzerland, Departmentof Oral Surgery and Stomatology, University ofBern, Bern, SwitzerlandBernd Stadlinger, Clinic of Cranio-Maxillofacialand Oral Surgery, University of Z€urich, Z€urich,SwitzerlandHendrik Terheyden, Department of Oral &Maxillofacial Surgery, Red Cross Hospital, Kassel,Germany

Corresponding author:Dieter D. BosshardtRobert K. Schenk Laboratory of Oral HistologySchool of Dental Medicine, University of BernFreiburgstrasse 7CH-3010 Bern, SwitzerlandTel.: +41 31 632 86 05Fax: +41 31 632 49 15e-mail: dieter.bosshardt@zmk.unibe.ch

Key words: cell communication, cementoblast, cementogenesis, cementum, enamel matrix

proteins, growth factors, periodontal regeneration, wound healing

Abstract

Background: Although regenerative treatment options are available, periodontal regeneration is

still regarded as insufficient and unpredictable.

Aim: This review article provides scientific background information on the animated 3D film Cell-

to-Cell Communication – Periodontal Regeneration.

Results: Periodontal regeneration is understood as a recapitulation of embryonic mechanisms.

Therefore, a thorough understanding of cellular and molecular mechanisms regulating normal

tooth root development is imperative to improve existing and develop new periodontal

regenerative therapies. However, compared to tooth crown and earlier stages of tooth

development, much less is known about the development of the tooth root. The formation of root

cementum is considered the critical element in periodontal regeneration. Therefore, much research

in recent years has focused on the origin and differentiation of cementoblasts. Evidence is

accumulating that the Hertwig’s epithelial root sheath (HERS) has a pivotal role in root formation

and cementogenesis. Traditionally, ectomesenchymal cells in the dental follicle were thought to

differentiate into cementoblasts. According to an alternative theory, however, cementoblasts

originate from the HERS. What happens when the periodontal attachment system is traumatically

compromised? Minor mechanical insults to the periodontium may spontaneously heal, and the

tissues can structurally and functionally be restored. But what happens to the periodontium in case

of periodontitis, an infectious disease, after periodontal treatment? A non-regenerative treatment

of periodontitis normally results in periodontal repair (i.e., the formation of a long junctional

epithelium) rather than regeneration. Thus, a regenerative treatment is indicated to restore the

original architecture and function of the periodontium. Guided tissue regeneration or enamel

matrix proteins are such regenerative therapies, but further improvement is required. As remnants

of HERS persist as epithelial cell rests of Malassez in the periodontal ligament, these epithelial cells

are regarded as a stem cell niche that can give rise to new cementoblasts. Enamel matrix proteins

and members of the transforming growth factor beta (TGF-ß) superfamily have been implicated in

cementoblast differentiation.

Conclusion: A better knowledge of cell-to-cell communication leading to cementoblast

differentiation may be used to develop improved regenerative therapies to reconstitute

periodontal tissues that were lost due to periodontitis.

In the film and in the present review, peri-

odontal regeneration is understood as a reca-

pitulation of embryonic mechanisms leading

to the development of the periodontium.

Development, homeostasis, pathology, repair,

and regeneration are the result of coordinated

interactions between cells that communicate

with each other. Communication can occur

via direct cell-to-cell contact or by secreted

molecules that bind to receptors expressed on

the cell surface of effector or target cells.

Besides a paracrine mode of cell communica-

tion, autocrine and endocrine mechanisms are

essential as well and add to the complexity of

biologic systems. Understanding cell-to-cell

communication during tooth development

has two important aims: (i) It forms the basis

for a better understanding of cell communica-

tions leading to periodontal regeneration. (ii)

It is the requisite know-how for the improve-

ment of existing therapies and development of

new periodontal regenerative strategies.

The periodontium

The periodontium is a functional unit that

connects the tooth with its surrounding bone

Date:Accepted 04 December 2014

To cite this article:Bosshardt DD, Stadlinger B, Terheyden H. Cell-to-cellcommunication – periodontal regeneration.Clin. Oral Impl. Res. 00, 2014, 1–11doi: 10.1111/clr.12543

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1

and provides the peripheral defense mecha-

nism against infection (Nanci & Bosshardt

2006; Bosshardt 2010). It comprises the gin-

giva (marginal periodontium), root cemen-

tum, periodontal ligament, and the alveolar

bone proper (bundle bone) (Fig. 1). While the

gingiva and periodontal ligament are special-

ized soft connective tissues, cementum and

bone are mineralized tissues. Cementum,

periodontal ligament, and alveolar bone con-

stitute the tissues that attach the tooth to

the surrounding bone. Collagen fibers,

embedded in bone and cementum, span

across the periodontal ligament and thus pro-

vide a flexible connection between the tooth

and surrounding bone. The tooth is a unique

organ in our body. Unlike other organs or tis-

sues, it does not undergo physiologic remod-

eling. However, injury to the tooth and to

the periodontal tissues or infection may initi-

ate a process leading to loss of dental and

periodontal tissues. How much structure and

function of a tooth and its periodontal tissues

are impaired may depend on the type, sever-

ity, and duration of insult. Recovery may

result in repair or regeneration of lost tissues,

and function may therefore be restored par-

tially or completely, respectively. As the peri-

odontium is closely connected to and

develops with the tooth, an understanding of

tooth development is mandatory for a discus-

sion about periodontal regeneration.

Tooth development

Over 300 genes are associated with tooth

development (Thesleff 2006; http://bite-it.hel-

sinki.fi). The majority of these genes mediate

cellular communication. Tooth development

is a paramount example of cell-to-cell com-

munication between epithelial (ectodermal)

and ectomesenchymal cells. Reciprocal epi-

thelial–mesenchymal interactions drive tooth

development from the tooth bud to the cap

and bell stages (Fig. 2) (Thesleff et al. 1995;

Thesleff 2006; Lesot & Brook 2009). In the

late bell stage, histodifferentiation begins,

leading to the differentiation of hard tissue-

forming cells. While odontoblasts (Fig. 3)

differentiate from ectomesenchymal (neural

crest-derived) cells and form coronal dentin,

ameloblasts are of ectodermal origin and

produce enamel. Pre-ameloblasts and amelo-

blasts (Fig. 4) synthesize and secrete a mix-

ture of proteins that form the enamel matrix,

a template for enamel mineralization.

Enamel matrix proteins (EMPs) include

amelogenin, ameloblastin (also known as

amelin or sheathlin), amelotin, tuftelin, apin,

and enamelin. Alternative splicing and pro-

tein degradation add to the complexity of the

enamel matrix. Besides biomineralization,

EMPs have cell-signaling functions (Zeich-

ner-David 2001; Bosshardt 2008). Of particu-

lar interest is the notion that specific

amelogenin splice products may function as

potential epithelial–mesenchymal signaling

molecules, another example of cell-to-cell

communication (Bosshardt 2008).

Toward the end of tooth crown develop-

ment, epithelial cells from the enamel organ

grow apically and form the HERS, an epithe-

lial double cell layer indispensable for root

formation. In this way, epithelial–mesenchy-

mal interactions continue until completion

of root development (Huang & Chai 2012).

One result of these cell-to-cell interactions is

the differentiation of ectomesenchymal cells

into odontoblasts, which form the dentin of

the root. While the root grows apically, more

coronally located portions of the HERS

Fig. 1. Micrograph illustrating the periodontal ligament

(PL) with its collagen fiber bundles spanning between

the root covered with cementum (C) and the alveolar

bone (AB). D, dentin. Undecalcified ground section,

unstained and viewed under polarized light. (Reprinted

from Bosshardt & Sculean 2009 with permission).

Fig. 2. Each tooth develops via communication between ectodermal and ectomesenchymal cells from a tooth bud

to the cap stage followed by the bell stage. (Screenshot Cell-to-Cell Communication – Periodontal Regeneration).

Fig. 3. Odontoblasts, which differentiate from ectomesenchymal cells residing in the dental papilla, produce dentin.

Every odontoblast possesses a long cytoplasmic process that passes in a dentinal tubule trough the whole dentin

matrix layer. Scanning electron microscopic image, 45009. (Courtesy of eye of science).

2 | Clin. Oral Impl. Res. 0, 2014 / 1–11 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Bosshardt et al �Periodontal regeneration - communication of cells

disintegrate. Fragments of this disintegration

process constitute the epithelial cell rests of

Malassez (ERM) (Rincon et al. 2006), which

reside in the periodontal ligament throughout

life. The periodontal ligament arises from the

dental follicle, an ectomesenchymal tissue

encasing the developing tooth. Disintegration

of HERS exposes the dentin surface and

makes it accessible to other cells, for exam-

ple, dental follicle cells. This is the moment

when cementoblast differentiation starts and

cementum matrix begins to be deposited on

the exposed dentin surface.

While numerous signaling pathways and

transcription factors are involved in regulat-

ing tooth crown development (Zhang et al.

2005), little is known about the molecular

mechanisms of tooth root development. Ces-

sation of fibroblast growth factor (FGF) 10

(Yokohama-Tamaki et al. 2006), epidermal

growth factor (Fujiwara et al. 2009), and epi-

thelial bone morphogenetic protein (BMP)

(Yang et al. 2013) signaling appears to be

involved in the transition from crown to root

formation. Furthermore, Wnt/ß-catenin sig-

naling is significant to root formation (Zhang

et al. 2013).

Cementogenesis

Before cementoblasts commence to secrete

the cementum matrix, HERS cells have been

shown to express EMPs, which accumulate

on the surface of recently formed dentin

(Fig. 5) (Bosshardt & Nanci 2004). These

EMPs together with growth factors, such as

members of the TGF-ß superfamily, are held

responsible for triggering the differentiation

of precursor cells into cementoblasts, the

cementum-forming cells. The initial stages of

cementogenesis have been studied in human

teeth during the first half of root develop-

ment, which covers the development period

of acellular extrinsic fiber cementum (Boss-

hardt & Schroeder 1991a,b). The cemento-

blasts synthesize, secrete, and implant a

dense fringe of collagen fibers, the pre-

cementum, into the dentin matrix before

mineralization starts (Figs 6 and 7). After

implantation of this short fiber fringe, the

dentin matrix mineralizes followed by miner-

alization of pre-cementum (Figs 8–10). The

mineralized portions of the fibers inserting

into cementum are called Sharpey’s fibers.

On the opposite side of the developing peri-

odontal ligament, short collagenous fiber

stubs become embedded in the bone matrix

and constitute the Sharpey’s fibers of bone.

The fiber fringes implanted in root dentin

and in bone remain short until the tooth

approaches the occlusal plane. Elongation of

the short collagen fiber stubs from both the

root surface and bone results in the forma-

tion of the collagenous fiber network of the

periodontal ligament, which ensures attach-

ment of the tooth to the surrounding bone

(see Fig. 1).

The formation of root cementum is consid-

ered critical, because it is thought to directly

determine the extent and quality of the

regenerative outcome. To date, origin and dif-

ferentiation mechanisms of cementoblast pro-

genitors are unclear, and this is true for

development and regeneration. Although the

Fig. 4. Ameloblasts, which differentiate from the inner

cells of the ectodermal enamel organ, form enamel.

Scanning electron microscopic image, 35009. (Courtesy

of eye of science).

Fig. 5. Transmission electron micrograph illustrating

an immunocytochemical preparation with an anti-ame-

logenin antibody and the protein A-gold technique. At

the very beginning of cementogenesis, round organic

matrix deposits labeled with gold particles (arrows) are

randomly distributed along the dentin (D) surface where

they co-localize with the collagenous matrix of prece-

mentum (PC). (Reprinted from Bosshardt & Nanci 2004

with permission).

Fig. 6. Transmission electron micrograph illustrating

the initial attachment of the collagenous matrix of acel-

lular extrinsic fiber cementum to the predentin (PD)

matrix of the root in the region of the future dentino-

cemental junction (DCJ). (Reprinted from Bosshardt &

Selvig 1997 with permission).

Fig. 7. Collagen fibers become attached to the dentin of

the root, and their mineralization results in the forma-

tion of root cementum. (Screenshot Cell-to-Cell Com-

munication – Periodontal Regeneration.)

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 3 | Clin. Oral Impl. Res. 0, 2014 / 1–11

Bosshardt et al �Periodontal regeneration - communication of cells

periodontal ligament is known to harbor cells

expressing mesenchymal stem cell markers

such as STRO-1 and CD146 (Seo et al. 2004;

Trubiani et al. 2005; Bartold et al. 2006), the

stem cell population in the periodontal liga-

ment is heterogeneous, and their precise loca-

tions and the cell differentiation mechanisms

giving rise to new periodontal ligament fibro-

blasts, osteoblasts, and cementoblasts are

unknown (Bosshardt 2005; Bartold et al.

2006).

As the ability to identify and manipulate

stem cells is important in regenerative medi-

cine, particularly for the development of tissue

engineering-based therapies, more research is

needed to pinpoint periodontal stem cells, to

find out where these cells reside within the

periodontium, and determine cell markers to

identify and selectively isolate these mesen-

chymal stem cells. For an understanding of

regenerative processes and development of

improved and more predictable regenerative

therapies, an understanding of periodontal

development during root formation is manda-

tory. According to the classical and most

widely accepted theory, cementoblast precur-

sors originate from the dental follicle proper

(Ten Cate 1997; Cho & Garant 2000). Accord-

ing to an alternative theory, cementoblasts

originate from the HERS (MacNeil & Thomas

1993; Bosshardt 1994; Beck et al. 1995; Davi-

deau et al. 1995; Thomas 1995; Bosshardt &

Schroeder 1996; Webb et al. 1996; Bosshardt &

Nanci 1997, 1998, 2000, 2004; Bosshardt &

Selvig 1997; Bosshardt et al. 1998a; Terling

et al. 1998; Lezot et al. 2000; Bosshardt 2005).

This concept implies that an epithelial–mes-

enchymal transition (EMT) occurs during

tooth root development. EMT is a fundamen-

tal process in phylogenetic, embryonic, and

neoplastic development (Hay 2005; Thiery

et al. 2009). Not surprisingly, EMT is also the

mechanism used by cancer cells to disperse

throughout the body. Thus, an interesting and

important association of EMT exists between

normal development and tumor formation.

TGF-ß is commonly associated with EMT and

tumor invasion, emphasizing its important

role in cell-to-cell communication in both

healthy and pathologic conditions.

It is well accepted that periodontal regener-

ation is a recapitulation of tooth developmen-

tal processes. There is no doubt that some

cells originating from HERS persist in the

periodontal ligament as the ERM (Rincon

et al. 2006). For many years, it has been spec-

ulated as to what their role(s) may be. Appar-

ently, they respond to environmental

stimuli, such as inflammation, as do HERS

cells by the production of amelogenins. Cells

of the ERM can be induced to proliferate

both in vivo and in vitro, can form cysts and

tumors, and can express EMPs during peri-

odontal wound healing (Hasegawa et al.

2003) and as a response to chronic inflamma-

tion (Hamamoto et al. 1996). Other studies

reported that the ERM synthesized bone/

cementum-related proteins such as osteopon-

tin and bone sialoprotein (Hasegawa et al.

2003; Mouri et al. 2003; Mizuno et al. 2005;

Rincon et al. 2005a,b), as well as amelogenin

and amelin (Fong & Hammarstr€om 2000).

These data indicate a potential role in peri-

odontal regeneration. However, their precise

role in wound healing and regeneration is

still unclear. Taking into consideration the

proposed origin of cementoblasts from HERS,

the ERM may likewise be a source for new

cementoblasts. In a new hypothesis, it was

proposed that the ERM constitute a unique

stem cell niche within the periodontal

Fig. 8. Transmission electron micrograph illustrating at the mineralization front the entrance of the collagenous

periodontal ligament fibers (CF) into the acellular extrinsic fiber cementum (AEFC). CB, cementoblast. (Reprinted

from Bosshardt & Sculean 2009 with permission).

Fig. 9. The collagen fibers (CF) embedded in cementum (C) on the root surface continue as principal periodontal lig-

ament fibers into the periodontal ligament space. D, dentin. Scanning electron microscopic image, 15,0009. (Cour-

tesy of eye of science).

Fig. 10. Cementoblasts reside between the extruding

collagen fibers (CF) close to the root surface. AEFC,

acellular extrinsic fiber cementum. Scanning electron

microscopic image, periodontal ligament, 30009. (Cour-

tesy of eye of science).

4 | Clin. Oral Impl. Res. 0, 2014 / 1–11 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Bosshardt et al �Periodontal regeneration - communication of cells

ligament and contain a subpopulation of

stem cells undergoing EMT for periodontal

repair and regeneration (Bosshardt 2005). This

concept has initiated a series of studies sup-

porting the proposed EMT (Sonoyama et al.

2007; Akimoto et al. 2011; Kapferer et al.

2011; Xiong et al. 2012, 2013; Lee et al.

2014). As TGF-ß1 and FGF2 stimulate the

EMT of HERS (Chen et al. 2014), these two

growth factors are candidate molecules for

periodontal regeneration. Taken together, it

is reasonable to believe that the ERM is

important for predictable and successful peri-

odontal regeneration (Bosshardt 2005; Rincon

et al. 2006; Xiong et al. 2013).

Orthodontic tooth movement

The tooth attachment system may be chal-

lenged during orthodontic tooth movement.

It seems like a miracle that a tooth can be

moved through the bone, a mineralized tis-

sue, and stay intact. While bone resorption

and apposition are prerequisites for tooth

movement, the tooth is less prone to resorp-

tion when appropriate forces are applied. The

high physiologic turnover rate and tissue

architecture facilitate bone remodeling.

Under mechanical pressure, pro-inflamma-

tory signaling involving cytokines induces

the bone lining cells (passive osteoblasts) to

retract, and osteoclast precursors are

recruited and differentiate into bone-resorb-

ing osteoclasts. Osteoclasts bind via receptors

to ligands (e.g., osteopontin) exposed on the

denuded bone matrix surface. They excavate

a convex resorption cavity on the bone sur-

face, the Howship’s lacuna, a distinct mor-

phological sign of resorption. In the reversal

phase, bone apposition follows bone resorp-

tion. After the withdrawal of the osteoclasts,

osteoblasts deposit the bone matrix. Bone

resorption and apposition are tightly coupled.

Osteocytes have an important role in the reg-

ulation of bone remodeling (Bonewald 2011).

They respond to mechanical stimuli and pro-

duce sclerostin (SOST), a negative regulator

of bone formation (van Bezooijen et al. 2004).

This is just another great example of cell-

to-cell communication in the periodontium.

Depending on the magnitude and direction

of force applied, the compressed periodontal

ligament may focally become necrotic, and at

these sites, odontoclasts may resorb cemen-

tum and dentin on the tooth root (Fig. 11)

(Bosshardt et al. 1998b). Before and concomi-

tant with root resorption, macrophages and

multinucleated giant cells may be found in

the necrotic periodontal ligament, where they

remove necrotic tissue (Fig. 12) (Bosshardt

et al. 1998b). Compared to bone resorption,

root resorption is less pronounced, because the

tooth root is less accessible for resorbing cells

due to the avascularity of cementum and den-

tin. After the withdrawal of the odontoclast,

new collagen fibers become attached to the re-

sorbed root surface (Fig. 13), and mineraliza-

tion forms a cementum layer, which embeds

these fibers (Fig. 14). The events on the ten-

sion sites around teeth are different from sites

with compression. Bone resorption is lacking,

and accelerated bone formation occurs at sites

where the periodontal ligament is under ten-

sion (Di Domenico et al. 2012). But what hap-

pens when the periodontal attachment system

is severely compromised, for example, by

strong mechanical insult or infection?

Trauma

Trauma affects the periodontal ligament and

may lead to a cementum defect, which

weakens the attachment function. When the

periodontal ligament becomes compressed, a

focal necrosis may develop. Hemorrhage due

to blood vessel damage and fenestration

causes extravasation of blood cells (e.g.,

monocytes, neutrophils, erythrocytes, and

platelets) and blood plasma, followed by clot-

ting (Figs 15–17). Macrophages migrate from

the margins of the intact periodontal liga-

ment into the damaged area and remove

necrotic tissue fragments. Macrophages are

assisted by multinucleated giant cells, which

are actually fused macrophages (Fig. 18). In

humans, the first multinucleated giant cells

can be observed as early as 1 week after the

start of a mechanical trauma (Bosshardt et al.

1998b). In the areas where macrophages and

multinucleated giant cells are engaged in

cleaning up the necrotic tissue, odontoclasts

are frequently found on the root surface,

which they superficially resorb (Fig. 19). The

resorption of cementum leads to loss of

attachment function.

Cells from the monocyte/macrophage line-

age have important functions in necrotic tis-

sue removal, wound healing, and regeneration.

Macrophages (Fig. 20) are monocyte-derived

myeloid cells, constitute a heterogeneous cell

population, and display remarkable plasticity.

They can change their phenotype depending

on the local microenvironment. Polarized

macrophages are commonly known as either

an M1 or an M2 phenotype. The M1 pheno-

type is the “classically activated” proinflam-

matory macrophage, whereas the M2

phenotype is a “wound healing” macrophage

(Mosser & Edwards 2008). Macrophages can

release signaling molecules for angiogenesis

and recruitment of progenitor and stem cells

involved in tissue regeneration (Brown et al.

2012). Thus, macrophages have many func-

tions and play important roles in cell-to-cell

communication when necrotic tissue removal

is completed and switches to regeneration.

How the differentiation of cementoblasts,

periodontal ligament fibroblasts, and osteo-

blasts, all cells needed to restore the attach-

ment function, is regulated is still not clear

and is therefore a matter of intense investiga-

tion around the world. What is more clear is

that a defect on the tooth root can spontane-

ously heal, a process called surface repair. If

the damaged area along the root surface is

larger than a few millimeters, the periodontal

ligament cells may be unable to migrate fast

enough from the adjacent intact periodontal

ligament tissue. Instead, osteoblasts from the

alveolar bone, which is only 200 lm away

from the root, are activated. Signaling

molecules involved in differentiation and

activation of osteoblasts include growth fac-

tors such as BMPs. This way, bone can grow

into the area of the not yet regenerated

periodontal ligament. While approaching the

root surface, bone can even grow further

Fig. 11. Light micrograph illustrating an advanced stage

of root resorption at a pressure site 3 weeks after the

onset of orthodontic tooth movement. Small blood ves-

sels, extravasated erythrocytes, and multinucleated giant

cells (MNGC) are seen in the damaged periodontal liga-

ment (PL), whereas odontoclasts (OC) are found on the

resorbed root surface. C, cementum; D, dentin. (Rep-

rinted from Bosshardt et al. 1998b with permission).

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 5 | Clin. Oral Impl. Res. 0, 2014 / 1–11

Bosshardt et al �Periodontal regeneration - communication of cells

into the resorption cavities formed by

odontoclasts on the root surface. The more

dentin is lost by resorptive activity, the more

bone can expand into the root and become

firmly connected to the dentin matrix. Bone

now takes over tissue remodeling and further

invades into the root. This process is called

replacement resorption and results in ankylo-

sis, which can be diagnosed radiologically,

clinically, and histologically (Fig. 21).

Periodontitis

Periodontal disease, one of the most impor-

tant oral diseases that contribute to the bur-

den of chronic disease, is highly prevalent

and represents a major health problem world-

wide (Petersen & Ogawa 2012). Furthermore,

an association exists between periodontal

diseases and several systemic conditions

(Cullinan & Seymour 2013). Thus, prevalence

and associations with systemic diseases

emphasize the need to prevent and treat peri-

odontal diseases. Gingivitis and periodontitis

are infectious periodontal diseases, and they

may be acute or chronic (Lindhe et al. 2008).

Periodontitis represents a more advanced

form of infection that causes destruction of

the tooth-supporting periodontal tissues. Var-

ious cytokines and proteolytic enzymes

released by immune cells in response to bac-

terial infection can cause extensive degrada-

tion of soft connective tissue (Ebersole et al.

2013), another example of cell-to-cell com-

munication. This leads to inflammatory root

resorption and eventually large-scale loss of

connective tissue fibers. For a detailed insight

into the cellular and molecular events associ-

ated with the inflammatory reaction caused

by a periodontal infection, the readers are

referred to a recent comprehensive review

(Terheyden et al. 2014).

Inflammation, pocket formation, and bone

resorption are the hallmarks of periodontitis.

Resorption of bone is performed by osteo-

clasts. The control of osteoclast formation is

another excellent example of cell-to-cell com-

munication. About 30 years ago, it was sug-

gested that the osteoblast lineage controls

the formation of osteoclasts. Fifteen years

later, the molecular mechanisms for the cell-

to-cell interaction regulating bone resorption

were discovered (Martin 2013). Normal bone

remodeling depends on a delicate balance

between bone formation and resorption. Bone

resorption is regulated by the RANK/

RANKL/OPG system. Receptor activator of

nuclear factor kappa-B (RANK) and its ligand

RANKL are members of the tumor necrosis

factor (TNF) ligand and receptor families.

RANKL is expressed as a membrane-bound

or secreted ligand by osteoblasts and certain

fibroblasts, whereas RANK is expressed by

osteoclast precursors and mature osteoclasts.

The binding of RANK to RANKL induces

Fig. 12. Light microscopic image showing multinucleated giant cells (MNGC) and blood vessels (BV) at a pressure

site in the periodontal ligament. C, cementum; D, dentin. (Courtesy of DD Bosshardt).

Fig. 13. Transmission electron micrograph illustrating

the new attachment of collagen fibers (CF) to the re-

sorbed dentin (D) of the tooth root. (Reprinted from

Bosshardt 1994 with permission).

Fig. 14. Transmission electron micrograph illustrating

the electron-dense, initial layer of mineralized cemen-

tum (C) after new collagen fiber (CF) attachment to the

resorbed dentin (D) of the root. (Courtesy of DD Boss-

hardt).

Fig. 15. Transmission electron micrograph illustrating a compressed region of the periodontal ligament 3 weeks

after the onset of orthodontic tooth movement. Extravasated erythrocytes (EC), platelets (P), and blood plasma are

present in the extravascular space close to a fenestration (arrow) in the endothelial cell lining of a blood vessel (BV).

(Reprinted from Bosshardt et al. 1998b with permission).

6 | Clin. Oral Impl. Res. 0, 2014 / 1–11 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Bosshardt et al �Periodontal regeneration - communication of cells

osteoclast differentiation and activity and

regulates their survival. Osteoprotegerin

(OPG), however, which is produced by osteo-

genic cells and certain fibroblasts, is a soluble

decoy receptor for RANKL that competes for

binding to RANK. Thus, OPG is a natural

inhibitor of osteoclast differentiation and

activation. Any interference with this system

can shift the balance between bone apposi-

tion and resorption. The expression of macro-

phage colony-stimulating factor (M-CSF)

plays an essential role in this regulatory sys-

tem. Furthermore, it has been shown that a

number of pro-inflammatory cytokines and

growth factors, in particular interleukin 1

(IL-1), interleukin 17 (IL-17), and TNF-a, reg-

ulate the expression of RANKL and OPG.

The immune system modifies the balance

between bone formation and resorption in a

complex process involving T-cells but also B-

cells, dendritic cells, and cytokines. By the

expression of RANKL on activated T-cells and

the expression of RANK on osteoclast precur-

sors and mature osteoclasts, these cells can

directly influence bone resorption (Hofbauer

& Heufelder 2001; Goldring 2003; Clowes

et al. 2005). The discovery of this important

cross talk between bone cells and the immune

system has opened possibilities for new thera-

peutic applications against bone resorption.

For instance, denosumab, a RANKL-neutraliz-

ing antibody, is in clinical use in patients with

osteoporosis, rheumatoid arthritis, and tumor-

related osteolysis (Kearns et al. 2008). This is

an impressive example illustrating that deci-

phering cell-to-cell communication can lead

to the development of new therapies.

The irreversible breakdown of the support-

ing periodontal tissues progressively leads to

tooth loosening and eventually tooth loss.

Tissue breakdown may be retarded by appro-

priate therapeutic measures, but it cannot be

reversed. Fortunately, research has provided

evidence that in most situations, chronic

periodontal diseases can be treated (Green-

well 2001). Periodontally involved teeth

have a good chance of survival, provided that

Fig. 16. Transmission electron micrograph illustrating a compressed region of the periodontal ligament 3 weeks

after the onset of orthodontic toot movement. Platelets (P) are present in the lumen of a blood vessel. (Reprinted

from Bosshardt et al. 1998b with permission).

Fig. 17. Transmission electron micrograph illustrating a compressed region of the periodontal ligament 3 weeks

after the onset of orthodontic toot movement. Erythrocytes (EC) together with platelets (P), cross-linked fibrin (F),

and blood plasma form an extravascular blood clot. (Reprinted from Bosshardt et al. 1998b with permission).

Fig. 18. Transmission electron micrograph illustrating a compressed region of the periodontal ligament 5 weeks

after the onset of orthodontic toot movement. A large multinucleated giant cell is seen scavenging debris of the

necrotic periodontal ligament. (Courtesy of DD Bosshardt).

Fig. 19. Light microscopic image illustrating root resorp-

tion in the region of the compressed periodontal ligament

5 weeks after the onset of orthodontic tooth movement.

Note the TRAP-positive odontoclasts (OC) on the root

surface and the TRAP-positive multinucleated giant cells

(MNGC) in the damaged periodontal ligament (PL). C,

cementum; D, dentin. TRAP = tartrate-resistant acid

phosphatase. (Courtesy of DD Bosshardt).

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 7 | Clin. Oral Impl. Res. 0, 2014 / 1–11

Bosshardt et al �Periodontal regeneration - communication of cells

therapy, patient compliance, and mainte-

nance care are appropriate (Greenwell 2001).

There is a broad range of treatment options

available, but only few can be regarded as

regenerative procedures (Bosshardt & Sculean

2009). Results from well-designed, well-con-

trolled, and well-conducted animal studies

have shown that periodontal regeneration is

possible, yet histological data from humans

are very rare (Bosshardt & Sculean 2009).

Unfortunately, regenerative techniques are

very unpredictable and result in only partial

regeneration at best (Bartold et al. 2000;

Wang et al. 2005; Zohar & Tenenbaum 2005;

Bosshardt & Sculean 2009). After conven-

tional periodontal therapy, some periodontal

repair may occur. However, cementum regen-

eration and formation of a new connective

tissue attachment to the root require a peri-

odontal regenerative therapy. To develop

effective regenerative therapies, a thorough

understanding of cellular and molecular

mechanisms regulating normal tooth root

development is imperative.

Periodontal wound healing andregeneration

Any surgical periodontal intervention creates

a wound. After flap closure, the wound healing

cascade starts. The healing events are very

well documented for skin wounds (Nauta

et al. 2011), but much less is known about

wound healing in the oral cavity. Wound heal-

ing comprises four overlapping phases: hemo-

stasis, inflammation, proliferation, and tissue

remodeling (Guo & DiPietro 2010). Undis-

turbed and rapid wound healing are prerequi-

sites for regeneration to occur. However,

biologic and technical complications are asso-

ciated with periodontal wound healing and

regeneration (Bosshardt 2008; Bosshardt &

Sculean 2009). Important for periodontal

Fig. 20. Transmission electron micrograph illustrating a compressed region of the periodontal ligament 3 weeks

after the onset of orthodontic toot movement. A macrophage is seen in the damaged periodontal ligament. (Cour-

tesy of DD Bosshardt).

Fig. 21. Light microscopic image illustrating an anky-

losed human tooth. The alveolar bone (AB) is fused

with the cementum (C) on the root surface, while the

periodontal ligament does not exist anymore. D, dentin.

(Courtesy of DD Bosshardt).

Fig. 22. Light micrograph illustrating formation of a

long junctional epithelium (LJE) ending at the coronal

termination of regenerated cementum (C). D, dentin.

(Reprinted from Bosshardt & Sculean 2009 with permis-

sion).

Fig. 23. Schematic drawing illustrating the principle of

guided tissue regeneration. A barrier membrane is used

to form a secluded space with the aim to prevent the api-

cal growth of gingival cells and allow cells from the peri-

odontal ligament and alveolar bone to repopulate the

space under the membrane. (Courtesy of DD Bosshardt).

8 | Clin. Oral Impl. Res. 0, 2014 / 1–11 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Bosshardt et al �Periodontal regeneration - communication of cells

regeneration is stabilization of the blood clot

(Wikesj€o & Selvig 1999). Platelets synthesize

and release a variety of cytokines and growth

factors, such as platelet-derived growth factors

(PDGF-AB, PDGF-BB), TGF-ß, insulin-like

growth factor I (IGF-I), basic fibroblast growth

factor (FGF-2), vascular endothelial growth

factor (VEGF), and endothelial growth factor

(EGF) (Barrientos et al. 2008; Nurden 2011).

All of these signaling molecules have func-

tions in wound healing and regeneration. Fur-

thermore, platelet-rich plasma contains

fibrinogen, which is cleaved into fibrin to

accelerate wound healing. A recent review

provides more insight into the functions of

various cells and molecules on soft tissue

wound healing in the oral cavity (Sculean

et al. 2014).

Wound healing after a non-regenerative

periodontal therapy generally results in the

formation of a long junctional epithelium

(Fig. 22). This process is called periodontal

repair. The difference from periodontal regen-

eration is the lack of a new connective tissue

attachment to the root. No new cementum

with inserting collagen fibers can form on a

root covered by epithelial cells. There are,

however, techniques and devices available,

such as barrier membranes (guided tissue

regeneration; GTR) (Fig. 23) and bone grafting

materials (Fig. 24), that can provide a favor-

able environment for undisturbed wound

healing and periodontal regeneration (Fig. 25).

While preventing the apical growth of epithe-

lial cells, which leads to the formation of a

long junctional epithelium, these biomateri-

als stabilize the blood clot and favor the

growth of periodontal ligament into the

defect area. This is of great importance,

because the progenitor and stem cells capable

of regenerating a periodontal attachment

apparatus reside in the periodontal ligament

(Karring et al. 1993).

Another approach is to recapitulate embry-

onic mechanisms of tooth development. As

EMPs are implicated in cementogenesis dur-

ing root development, these molecules are

potent candidate molecules to stimulate peri-

odontal regeneration. Indeed, EMPs are avail-

able under the brand name Emdogain�

(Straumann) and have been in clinical use for

almost two decades to treat intrabony peri-

odontal defects, for root coverage procedures,

and for tooth replantation. Emdogain� con-

sists of an enamel matrix derivative, water,

and a carrier, propylene glycol alginate. When

topically applied, EMPs precipitate on the

root surface after scaling and root planing

(Fig. 26), (Miron et al. 2012) promote wound

healing, restrict epithelial downgrowth, and

support regeneration of the periodontal

attachment apparatus comprising cementum,

periodontal ligament, and bone. A large body

of in vitro data supports the beneficial effects

of EMPs to enhance periodontal regeneration

(Bosshardt 2008; Lyngstadaas et al. 2009; Scu-

lean et al. 2011; Grandin et al. 2012).

Acknowledgements: The authors

would like to thank Dr. Alexander Ammann

as the project manager of the previously

mentioned film project and Dr. Marko

Reschke and Mr. Matthias Gauer for the

artwork and technical production of the film.

The project was realized under the

economical responsibility of Quintessence

Publishing, Berlin, Germany. The film

project was supported by Institut Straumann.

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