Adv in Cementum Devt[1]

3 ____________________________________________________________________________

Advances in Defining Regulators of CementumDevelopment and Periodontal Regeneration

Brian L. Foster,* Tracy E. Popowics,{ Hanson K. Fong,{ andMartha J. Somerman*,{

*Department of Periodontics, School of Dentistry

University of Washington, Seattle, Washington 98195{Department of Oral Biology, School of Dentistry

University of Washington, Seattle, Washington 98195{Department of Materials Science and Engineering

University of Washington, Seattle, Washington 98195

I. I

Curre

Copy

ntroduction

II. Q
uestion 1. What Are the Unknowns That Must Be Considered in Order to Replicate the
Enamel (Crown) and How Do the Proteins Involved in Crown Development Relate to

Root Development?

A
. E
nt T

right

namel Structure

B
. E namel Biomineralization: Role of Proteins
C
. F uture Prospects for Enamel Regeneration
III. Q
uestion 2. What Do We Know About the Cells Required for Periodontal Development
and Regeneration?

A
. D evelopmental Cells
B
. D erivation of Cementum: Competing Theories of Cementoblast Origin
C
. D iVerences Between Cementoblasts and Osteoblasts
D
. T ooth Stem Cell Populations
IV. Q
uestion 3. What Genes and Associated Proteins Are Important for Root/Periodontal
Tissue Formation?

A
. F actors Associated with the Putative Epithelial Niche (HERS and ERM)
and Surrounding Mesenchyme

B
. B one Morphogenetic Proteins
C
. P eriostin and Nuclear Factor I‐C/CAAT Box Transcription Factor
D
. R egulators of Phosphate and Pyrophosphate Metabolism
E
. F actors Known to Regulate Osteoprogenitor Cells and Osteoblasts
F
. E merging and Other Factors to Consider
V. C
onclusions and Future Directions
A
cknowledgments
R
eferences
Substantial advancements havebeenmade in defining the cells andmolecular

signals that guide tooth crown morphogenesis and development. As a result,

very encouragingprogress has beenmade in regenerating crown tissuesbyusing

opics in Developmental Biology, Vol. 78 0070-2153/07 $35.002007, Elsevier Inc. All rights reserved. 47 DOI: 10.1016/S0070-2153(06)78003-6

48 Foster et al.

dental stem cells and recombining epithelial andmesenchymal tissues of specific

developmental ages.Todate, attempts to regenerate a complete tooth, including

the critical periodontal tissues of the tooth root, have not been successful. This

may be in part due to a lesser degree of understanding of the events leading to

the initiation and development of root and periodontal tissues. Controversies

still exist regarding the formation of periodontal tissues, including the origins

and contributions of cells, the cues that direct root development, and the

potential of these factors to direct regeneration of periodontal tissueswhen they

are lost to disease.

In recent years, great strides have been made in beginning to identify and

characterize factors contributing to formation of the root and surrounding

tissues, that is, cementum, periodontal ligament, and alveolar bone. This

review focuses on the most exciting and important developments over the

last 5 years toward defining the regulators of tooth root and periodontal

tissue development, with special focus on cementogenesis and the potential

for applying this knowledge toward developing regenerative therapies. Cells,

genes, and proteins regulating root development are reviewed in a question‐answer format in order to highlight areas of progress as well as areas of

remaining uncertainty that warrant further study. � 2007, Elsevier Inc.

I. Introduction

During the last decade, we have gained substantial insights into the mechanisms

and factors controlling formation of many organs and tissues and with this, new

ideas on how to regenerate tissues lost as a consequence of pathologies, injuries,

and genetic disorders. These insights, based on new technologies and on the

exponential growth in defining the factors/genes/proteins regulating tissue and

organ development, have allowed us to enjoy more rapid discoveries than in the

past. Technological advances have resulted in increased eVorts to develop im-

provements in existing therapies targeted at replacement of lost tissues/organs/

body parts. An area of focus has been the oral cavity, with improvements seen in

(1) materials used to restore decayed, damaged tooth structure; (2) prosthetic

devices to replace missing teeth—full dentures, partial dentures, bridges, and

implants; and (3) materials/agents used to regenerate periodontal tissues—for

example, root cementum, periodontal ligament attachment, and alveolar bone.

In fact, with regard to developing clinical products that promote and/or protect

against bone loss, some of the newer products (on the market within the last

3 years) were first appreciated for their role in regulating key events during

formation and diVerentiation of hard tissues, including teeth. These include

bone morphogenetic protein [(rhBMP‐2): INFUSE; Medtronic Sofamor

Danek, Minneapolis, MN)] approved for clinical use in open fracture of long

bones, nonunions and vertebral arthrodesis, and parathyroid hormone (Fortio;

3. Regeneration of the Periodontium 49

teriparatide (rhDNA origin) injection contains human PTH (1‐34), Eli Lilly &

Co.) given intermittently to promote bone formation in individuals with severe

osteoporosis and in whom antiresorptive therapies have proven insuYcient.

To date, attempts to regenerate a complete tooth—crown, root, PDL,

bone—have not been successful; however, progress has been made in regen-

erating crown tissues. Because of the parallel of epithelial‐mesenchymal

(E‐M) signaling in crown formation, that is, ameloblasts‐enamel; odontoblasts‐dentin, withE‐Msignaling in other tissues during development, rodentmodels of

tooth crown development have been studied extensively, resulting in a wealth of

information as to the cells/factors and events controlling crown development

(Chai and Slavkin, 2003; Fong et al., 2005; ThesleV, 2003; ThesleV andMikkola,

2002; Zhang et al., 2005). Yet, there is still more information needed in order to

mimic enamel‐dental formation as a way to restore lost tissue structure. What is

known and was first realized decades ago is that appropriately timed mixing of

cells obtained from tooth epithelium with tooth mesenchyme simulates cell

diVerentiation toward ameloblasts and odontoblasts with subsequent crown

formation (the development of tooth germs in tissue culture, 1965; Duailibi

et al., 2004; Harada et al., 1999; Huggins et al., 1934; Kollar and Baird, 1969,

1970a,b; Kollar and Fisher, 1980;Mina and Kollar, 1987; Nieminen et al., 1998;

Ohazama et al., 2004b; Tucker and Sharpe, 2004; Young et al., 2002).

In contrast, the events/factors leading to formation of the root and surrounding

tissues, that is, cementum, alveolar bone and a functional periodontal ligament

(PDL) are just beginning to unfold.

This review focuses on the most exciting developments over the last 5 years

toward defining the regulators of root development, that is, cementogenesis

and the significance of this knowledge toward developing therapies targeted

at regeneration of a whole tooth and surrounding support structures.

We recognize that modulators of PDL and bone formation are key for root

formation and thus at times address these tissues, but the emphasis for this

review is on cementum. Further, based on the recent emphasis on defining the

possible role of epithelial‐derived factors during root development, a discussion

on the enamel‐associated factors produced by ameloblasts, and their known

and putative roles in formation of enamel and cementum, are discussed.

As the knowledge of factors that influence development of the period-

ontium increases in coming years, developing an eVective platform for the

delivery of these known factors will become increasingly important for pur-

poses of tissue regeneration. In the areas of drug delivery and tissue engi-

neering, advances have been made in the development of materials that can

serve as a vehicle to deliver proteins/genes/cells in vivo. Agents identified with

regenerative potential may then be partnered with delivery systems to local-

ize and regulate release of cells and factors at sites of repair/regeneration of

lost periodontal structures. Much exciting work has been done to advance

the design and fabrication of delivery systems, and several excellent reviews

Development

Diseased

Enamelbiomineralization

(#1)

Cells(#2) Genes/

proteins(#3)

Deliverysystem

Healthy

Figure 1 Progression of root development and regeneration. The tooth root develops as a

result of complex interactions of cells, signals, and matrix proteins, now just beginning to be

understood. The question‐answer format used in this review addresses recent progress in

defining key modulators of root development, and defines areas warranting further study.

Therapies targeted at regenerating the whole tooth will necessarily incorporate factors relating

to crown development and possibilities for enamel regeneration (Question 1), as well as cells

(Question 2) and genes/proteins (Question 3) that regulate periodontal development. Lastly,

cells and factors to be used in regenerative therapies should be partnered with eVective delivery

systems that serve as a scaVold for cells and/or function in controlled release of bioactive factors

to the local area.

50 Foster et al.

and primary publications may be consulted for detailed information on this

work (Abukawa et al., 2006; Bartold et al., 2006b; Jin et al., 2003; Nakahara,

2006; Taba et al., 2005).

A question‐answer format has been used to address progress in ascertain-

ing the key modulators of root development over the past 5 years, as well

as to recognize areas of remaining uncertainty that warrant further study.

A model visualizing the progression, from the stage of initiation of root

development and from a diseased periodontal state to a functional tooth is

shown in Fig. 1 to visually demonstrate the questions being posed.

1. What are the unknowns that must be considered in order to replicate

the enamel (crown) and how do the proteins involved in crown

development relate to root development?

2. What is known about the cells required for development and regeneration

of cementum?

3. What are the genes and associated proteins required for root development

and regeneration?


II. Question 1. What Are the Unknowns That Must BeConsidered in Order to Replicate the Enamel (Crown) andHow Do the Proteins Involved in Crown DevelopmentRelate to Root Development?

Enamel is the hardest biological tissue in the body. Although the primary

component of enamel, hydroxyapatite (HAP), does not compare favorably

with most known structural ceramics in terms of mechanical properties, it

exhibits remarkable durability. The key to enamel’s durability despite re-

peated attrition in the bacteria‐laden environment of the oral cavity lies in its

intricate microstructure; hence, regenerative strategies with the aim of suc-

cessful replication of the crown must involve not only the chemical makeup

of enamel, but the structural makeup as well. Current materials engineer-

ing technology has yet to find a way to fabricate the complex 3‐D enamel

structure. To complicate matters, mature enamel is a nonliving tissue, as the

ameloblasts that synthesize enamel matrix are lost on tooth eruption. A key

to enhancing progress toward regenerating enamel includes understanding

the cellular and molecular mechanisms regulating formation of this tissue.

The following discussion focuses on our current understanding of enamel

structure as it relates to mechanical functions, and on the genes/proteins

regulating enamel biomineralization. Also discussed are future approaches

to consider for designing regenerative enamel.

A. Enamel Structure

The organization of enamel can be imagined as a hierarchical structure,

starting at the smallest scale with HAP crystals approximately 50‐nm wide.

These crystals are bundled into a few micrometers wide which are referred

to as enamel rods or prisms, representing the next scale of hierarchy. The

interweaving of enamel rods builds the bulk of enamel tissue. The crystal

rod/interrod organization has been investigated carefully, and beautiful,

illustrative images can be found in textbooks such as Ten Cate’s Oral Histo-

logy (Nanci, 2003). Increasing evidence suggests that orientation and decus-

sation of enamel rods are important properties for preserving the

mechanical integrity of mature enamel (Marshall et al., 2001; Xu et al.,

1998). For example, rod organization is important for preserving the overall

enamel structural integrity by directing microcracks traveling through the

dentin‐enamel junction (DEJ) into the dentin, where they are then arrested

(Imbeni et al., 2005). Despite the extensive body of data characterizing the

enamel structure, several questions remain regarding key structural details

52 Foster et al.

that must be elucidated in order to understand the development and regener-

ation potential for enamel. Two of these critical questions include: (1) What

are the directional changes in enamel rods extending from the DEJ to enamel

surface, and (2) How are interrods structurally related to enamel rods?

Answering these questions is essential to achieve an adequate understanding

of the mechanical properties of enamel, as well as to gain insight toward

regeneration of a functional crown.

B. Enamel Biomineralization: Role of Proteins

1. Amelogenin

Amelogenin is the most abundant and best characterized protein in develop-

ing enamel. The amino acid sequence of amelogenin protein is highly con-

served across many species, suggesting physiologic relevance and common

functional properties across species (Paine and Snead, 2005). Amelogenin’s

eVect on enamel development has been aptly studied in both in vivo and

in vitro systems. Several lines of evidence support proper self‐assembly of

amelogenin proteins is essential for facilitating directional nucleation

of hydroxyapatite minerals. Human phenotypes for the condition amelogen-

esis imperfecta (AI) demonstrate lack of or altered amelogenin, resulting

in inferior enamel characterized by hypoplasticity or hypomineralization,

and often associated with disorganized enamel rods (Gibson et al., 2001b,

2005; Wright et al., 2003). Likewise, a severe form of AI, similar to humans

with amelogenin defects, was observed in amelogenin knockout (KO) mice

(Gibson et al., 2001a).

The current understanding of amelogenin’s role in enamel mineralization

has come to light through characterization of enamel in situ and isolated

recombinant amelogenin, in normal and defective forms. The first indication

of amelogenin’s ability to self‐assemble and dictate mineral organization

came from investigations focused on analyzing developing mouse enamel

where arrays of nanospheres were observed to approximate the sides of

needle‐like HAP crystallites (Moradian‐Oldak et al., 1995; Robinson et al.,

1981). Later, atomic force microscopy (AFM) and dynamic light scattering

measurements on M‐180 (mouse full‐length) amelogenin revealed that the

protein assembled into 20 nm nanospheres (Moradian‐Oldak et al., 2000).

Furthermore, dynamic light scattering measurements performed on M‐180with altered C‐terminal and N‐terminal domains indicated disruption of self‐assembly, resulting in smaller nanospheres with a wider size distribution

(Moradian‐Oldak et al., 2000). These findings revealed that C‐ and

N‐terminal domains were essential for proper amelogenin self‐assembly.


When transgenic mice were developed to express the same altered C‐ or N‐terminal domains of amelogenin, similar disruption in the nanosphere self‐assembly was observed, resulting in disruption in crystal organization of the

mineral phase during the secretory stage of enamel formation (Paine et al.,

2001). The resulting mature enamel was found to be hypomineralized with

disorganized rods, a direct eVect of altered C‐ and N‐terminal domains

manifested in a disorganized mineral phase in the nucleation stage of enamel

formation (Paine et al., 2001).

Details of the interactions between amelogenin and mineral are still

emerging, however the evidence to date indicates that amelogenin has a

strong binding aYnity to HAP via the hydrophilic C‐terminal domain.

Several investigators have demonstrated controlled HAP growth in the

presence of amelogenin using in vitro systems (Beniash et al., 2005; Iijima

et al., 2002). In one example, HAP crystals with a long ribbonlike morphol-

ogy resulted from growth in the presence of amelogenin (Iijima et al., 2002).

Another solution precipitation experiment showed formation of needle‐likeHAP crystals when amelogenin was introduced into the system (Beniash

et al., 2005). In both of these examples, the long axis of the crystals was the

crystallographic [001] direction (normal to the crystallographic (001) plane),

similar to that found in physiological enamel HAP, suggesting amelogenins

bind to the crystal surface(s) perpendicular to the (001) plane, limiting the

growth direction in [001] only. Additional binding studies by Hablitz et al.

showed that when amelogenins were introduced to a composite that exposed

fluoroapatite and glass, both having hydrophilic surfaces, amelogenins

bound only to fluoroapatite (Habelitz et al., 2004). NMR studies further

revealed that the binding site was through the C‐terminal domain of amelo-

genin (Shaw et al., 2004).

2. Non‐amelogenin Proteins

Although present in minor amounts relative to amelogenin, additional

enamel matrix proteins (EMPs) identified in developing enamel have been

shown to play a role in regulation of crystal growth. These non‐amelogenin

EMPs include enamelin, ameloblastin, tuftelin, biglycan, decorin, and ame-

lotin. The specific roles of these proteins in influencing biomineralization of

enamel are not fully understood and are currently under active investigation

(Table I). In both humans and mice, an AI‐like phenotype is the result of

mutation or KO of genes associated with some of these proteins, suggesting

that they play an important role in biomineralization.

Mutations in the enamelin gene in humans and mice result in AI char-

acterized by hypoplastic enamel (Hu and Yamakoshi, 2003; Kim et al., 2005;

Table I Factors Found in Developing Enamel

Factor Cells/Tissues Function/Putative Function Models References

Amelogenin Ameloblasts, HERS,

odontoblasts, periodontal

tissues (see also Table II)

Directs hydroxyapatite crystal

habit during developmental stage

of enamel formation by assembling

into extracellular protein matrix in

which mineral nucleates. Amelogenin

is also considered a potential

signaling molecule in dentin and

cementum development (Table II)

Human X‐linked amelogenesis

imperfecta (AI) (reduction

or elimination of amelogenin

expression by X‐chromosome):

partially hypomineralized enamel

Beniash et al. (2005);

Gibson et al. (2001a,b),

(2005); Iijima et al. (2002);

Lagerstrom ‐Fermer and

Landegren (1995);

Paine et al. (2001);

Wright et al. (2003)

Transgenic mice: altered A domain

(AA 1‐42) and altered B domain

(AA 157‐173) of M‐180amelogenin—hypomineralization

of enamel in both cases

Amelogenin KO mice:

hypomineralized enamel

(Table II regarding root

resorption)

In vitro mineralization: mineral

morphology control—short

needlelike or long ribbonlike

crystal shapes depending on

mineralization condition

Leucine‐rich amelogenin

peptide (LRAP)

Ameloblasts—alternative

splice product of

amelogenin

Suggested functions include:

responsible for binding to

hydroxyapatite, and implicated

as a signaling molecule in

periodontal tissue formation

Transgenic LRAP: expressed in

amelogenin null mice did not

rescue hypomineralized enamel

Boabaid et al. (2004b);

Chen et al. (2003)

LRAP overexpression in mice:

enamel pitting; in vitro, aVects

genes associated with PDL cells

and cementoblasts, and some

studies suggest proliferative

eVects

Tyrosine‐rich amelogenin

peptide (TRAP)

Ameloblasts—cleavage

product of amelogenin

Byproduct of amelogenin generated

by protease; no known eVect on

enamel; Implicated as signaling

molecule in periodontal tissue

formation

TRAP overexpression in mice:

no eVect on enamel, regulates

cementoblast behavior in vitro

Paine et al. (2004);

Swanson et al. (2006)

Enamelin Ameloblasts Regulates mineralization of enamel,

but its role has not been determined

Enamelin mutation in humans:

hypoplastic enamel

Kim et al. (2005);

Masuya et al. (2005);

Rajpar et al. (2001)Enamelin mutation in mice:

hypoplastic enamel

Ameloblastin Ameloblasts, ERM

(see also Table II)

Acts as a repressor of amelogenin,

limits ameloblast proliferation,

may regulate crystal nucleation

(see also Table II)

Ameloblastin KO mice:

Amelogenesis imperfecta

Ameloblastin overexpression in

mice: partially disrupted

enamel rod structure

Fukumoto et al. (2004,

2005); Paine et al. (2003)

Tuftelin Found concentrated in

dentin‐enamel

junction (DEJ)

May contribute to amelogenesis Tuftelin overexpression in mice:

disrupted rod/interrod

morphology

Luo et al. (2004)

Amelotin Ameloblasts Unknown Not reported Iwasaki et al. (2005)

Biglycan Bone, dentin, enamel Repressor of amelogenin Biglycan KO mice: transient

eVect included increased enamel

formation, interrod as primary

enamel structure while rod

structure was minimally affected

in newborns.

Adult teeth appeared normal

Goldberg et al.

(2002, 2005)

Decorin Bone, dentin, enamel Unknown Decorin KO mice: transient

eVect included decreased

enamel formation and

disorganized rod structure in

newborns. Adult teeth

appeared normal

Goldberg et al. (2005)

(Continued )

Dentin

sialophosphoprotein

(DSPP)

re‐secretoryameloblasts, DEJ

AVects DEJ morphology via

regulation of predentin formation

DSPP KO mice: irregular DEJ Sreenath et al. (2003)

Enamelysin (MMP‐20) entin, enamel Proteolytically breaks down enamel

proteins, e.g., amelogenin, in order

to facilitate mineral growth. MMP‐20is expressed in secretory and transition

stages of enamel development

MMP‐20 KO mice: disrupted rod

pattern, hypoplastic enamel

MMP‐20 mutation in mice: heavily

pigmented, hypoplastic enamel

Bartlett et al. (2004);

Bartlett et al. (2006);

Caterina et al.

(2002); Hu et al. (2002)

Kallikrein‐4 (KLK4) dontoblasts,

ameloblasts,

prostate

Proteolytically breaks down enamel

proteins, e.g., amelogenin, in order to

facilitate mineral growth. KLK4 is

expressed in transition and maturation

stages of enamel development

KLK4 mutation in human:

yellow‐brown discoloration, lower

mineral content in enamel

Hart et al. (2004);

Hu et al. (2002)

Table I Continued


P

D

O


Masuya et al., 2005; Rajpar et al., 2001). However, how enamelin interacts

with HAP mineral and/or the major matrix protein, amelogenin, remains

largely unknown.

Similarly, ameloblastin, whose expression by ameloblasts decreases from

secretory stage to maturation stage of amelogenesis, has been found to aVectmineralization. Ameloblastin KO mice were reported to develop hypocalci-

fied enamel with no recognizable rod structure (Fukumoto et al., 2004,

2005). In transgenic mice overexpressing (O/E) ameloblastin, Paine et al

observed disruption of the rod/interrod structure in localized regions of

enamel (Paine et al., 2003). Studies on ameloblastin null mice have also

suggested that the protein functions as a cell adhesion molecule and regula-

tor of cell growth (Fukumoto et al., 2004, 2005), however the specific role

and mechanism for ameloblastin in mineral formation remains unclear.

Tuftelin, another enamel protein with reported self‐assembly properties,

may be an important protein in enamel mineralization, but its physiological

function has yet to bewell characterized (Deutsch et al., 1998, 2002; Paine et al.,

1996, 1998). Tuftelin O/E in mice resulted in disruption of the enamel rod/

interrod structure, and the loss of the characteristic ribbonlike enamel crystallite

morphology within rods (Luo et al., 2004).

Other nonamelogenin proteins identified with enamel formation include

biglycan, decorin, and amelotin. Biglycan and decorin are leucine‐rich pro-

teoglycans, implicated in regulation of mineralized tissues. Loss of biglycan

or decorin expression in KO mice resulted in an increase or decrease in

enamel tissue formation, respectively (Goldberg et al., 2002). In both cases,

the mineral structure was initially disrupted, but recovered with maturation

of enamel. The reader is directed to Section IV.F for further information

on the proteoglycans decorin and biglycan, and their role in regulating

mineralized tissues of the tooth.

Amelotin, discovered and reported to be an ameloblast‐specific gene, hasbeen identified in humans and mice (Iwasaki et al., 2005). Expression of

amelotin mRNA was restricted to maturation stage ameloblasts in mice.

Amelotin’s potential role in enamel development is under investigation.

3. Proteases

While amelogenins are critical in the nucleation step of enamel biominerali-

zation, degradation of amelogenins is important for providing the space

for HAP mineral crystals to expand during the growth stage of amelogen-

esis. Two proteolytic enzymes, matrix metalloproteinase‐20 (MMP‐20) andkallikrein‐4 (KLK‐4), have been identified as enzymes required for breaking

down amelogenins (Bartlett et al., 1998; Fukae et al., 1998; Simmer et al.,

1998). MMP‐20, secreted into the enamel extracellular matrix by amelo-

blasts during the secretory stage, is responsible for the proteolytic cleavage

58 Foster et al.

of amelogenin protein into smaller fragments. A study by Ryu and co-

workers showed that incubation of MMP‐20 with recombinant porcine

amelogenin (rP172) produced the same cleaved amelogenin fragments that

are found in vivo (Ryu et al., 1999). KLK‐4, secreted during early maturation

stage, functions to further digest matrix proteins and cleavage products

incompletely digested by MMP‐20, facilitating nearly complete removal of

proteins from mature enamel (Simmer and Hu, 2002). The latest data using

in vitro models support the notion of diVerences in the way MMP‐20 and

KLK‐4 digest 32‐kDa enamelin. While MMP‐20 cleaved enamelin only after

it was deglycosylated, KLK‐4 readily cleaved enamelin into nine cleavage

products (Yamakoshi et al., 2006). Human phenotypes carrying mutated

MMP‐20 or KLK‐4 exhibit autosomal recessive hypomaturation AI (Hart

et al., 2004; Kim et al., 2005; Ozdemir et al., 2005). Likewise, hypoplastic

enamel with a disrupted rod pattern was reported in MMP‐20 null mice

(Caterina et al., 2002). The MMP‐20 null mice demonstrated that complete

elimination of EMPs from the enamel space failed to occur in the absence of

this proteolytic enzyme, resulting in limited space required for expansion of

the mineral phase.

C. Future Prospects for Enamel Regeneration

The knowledge accumulated to date on the structure and biomineralization

of enamel is abundant. However, there is much more to learn about these

two aspects of enamel before we can fully describe the structure–function

relationship of mature enamel and the processes involved in enamel forma-

tion. In terms of structure–function relationships, there is yet to be a clear

model describing the true 3‐D rod architecture throughout the tissue. The

ability to precisely describe directional changes of rods and their decussation

pattern from the DEJ to the crown surface is critical for understanding

the ability of the crown to distribute masticatory loads. From observation

of biological models, the building of the enamel structure has proven to be

a complex process. It requires an orchestration of protein–protein and

protein–mineral interactions that occur in a temporally and spatially co-

ordinated manner. Proper assembly and elimination of amelogenin has been

shown to be critical in nucleation and growth of the mineral phase during

formation of enamel. However, much is unknown with regard to the specific

functions of other proteins in the enamel biomineralization process. Fur-

thermore, emerging data have revealed that enamel proteins may serve a

critical role as signaling molecules in tooth root development (see Section

IV.A and Table II for details on the potential roles of enamel matrix proteins

in root formation). Continued investigations targeted at understanding the

detailed structure of enamel and the functions of individual EMPs, in enamel

Table II Factors Associated with the Putative Epithelial Niche (HERS and ERM) and Surrounding Mesenchyme


Msx2 (homeobox

containing

transcription

factor known to

play a role in

crown formation)

HERS (not in

apical

mesenchyme),

and present in

many other cell

types including

dental pulp and

PDL (limited)

Products from the mesenchymal cells in

the local region, e.g., BMPs, may

regulate HERS production of Msx2

and/or other factors. One outcome

of this interaction may be control of

root patterning

Msx2 KO: presence of

irregularly shaped molar

roots and increased

expression of periostin

Satokata et al.

(2000) ;

Yamamoto et al.

(2004a)

BMP‐2 and 4 (bone

morphogenetic

protein 2 and 4)

Apical mesenchyme/

follicle region

(not HERS)

BMP‐2/4 and possibly BMP‐3, alsofound in high concentrations in

the follicle region, regulate products

produced by the HERS cells; this

interaction controls growth and

morphogenesis of the root sheath,

and thus root patterning

BMP‐4 KO: arrested at

earlier stages of tooth

development, therefore

specific root defects are

unknown

Yamashiro et al.

(2003) ; Yamamoto

et al. (2004a)

FGF‐10 (fibroblast

growth factor 10)

Apical mesenchyme/

follicle region

(not HERS)

Continuous FGF‐10 expression by apical

mesenchyme maintains epithelial stem

cell population (as in continuously

erupting rodent incisors). Cessation of

FGF‐10 expression necessary for

transition to root formation in teeth of

limited eruption (e.g., rodent molars)

Mouse, FGF‐10 deficiency

and overexpression:

Deficiency: defect of

epithelial stem cell

(apical bud) compartment.

Harada et al. (1999),

(2002a,b);

Yokohama‐Tamaki

et al. (2006)

Overexpression: formation

of apical bud in mouse

molars, inhibiting HERS

formation and root

development

(Continued )

Ameloblastin Ameloblasts, HERS,

cementocytes

(low levels)

A known product of ameloblasts thought

to regulate enamel crystal size

Ameloblastin produced by HERS cells is

hypothesized by some to promote

acellular cementum formation

Ameloblastin KO: exhibits

an enamel phenotype but

no root deformities have

been reported

Simmer and Fincham

(1995); Zeichner ‐ David

et al. (2003)

Amelogenina Ameloblasts, HERS

region, odontoblasts,

PDL/cementum, and

possibly in other

tissues

A major protein of developing enamel and

a known product of ameloblasts involved

in regulating crystal structure. Suggested

functions in non ‐ epithelial tissues includeacting as a signaling molecule to regulate

diVerentiation of odontoblasts and

cementoblasts b. Hatakeyama et al. (2003,

2006) suggest that amelogenin acts to

protect the root from osteoclast‐mediated

root resorption

Amelogenin KO: exhibits

defective, chalky enamel

similar to that observed in

humans with amelogenesis

imperfecta (Gibson et al. ,

2001a). After root formation

is completed, root resorption

is enhanced in association

with osteoclasts and

cementicles in the

periodontal region

Boabaid et al. (2004b);

Bosshardt and Nanci

(2004); Bosshardt (2005);

Gibson et al. (2001b);

Hatakeyama et al. (2003);

Hatakeyama et al. (2006);

Nebgen et al. (1999);

Shimizu et al. (2005);

Veis et al. (2000);

Viswanathan et al. (2003)

Shh (sonic

hedgehog)

HERS, dental

mesenchyme, inner

enamel epithelium,

enamel knot

Involved in epithelial‐mesenchymal

interactions during tooth morphogenesis.

May contribute to root elongation through

signaling with Ptc1 and Gli1 genes and

proliferation of dental mesenchyme

Shh null mice: not viable

Ptc1 mes mutants: reduced

proliferation of mesenchyme

adjacent to HERS and

shorter roots

Nakatomi et al. (2006)

Table II Continued


IGF ‐1 (insulin ‐likegrowth factor ‐ I)

HERS May contribute to elongation of the HERS,

IGF receptors are present in vivo , and

elongation of HERS/increased cell

proliferation occurred in the outer

epithelial layer when IGF was added in vitro

IGF ‐ 1 localized to HERS in

5‐ day‐ old mice. In vitro

experiments supported a

role for IGF ‐ 1 in regulatingmitotic activity in HERS cells

Fujiwara et al. (2005)

OPN, BMP‐ 2,ameloblastin

Epithelial cell rests

of Malassez (ERM)

May assist in repair of cementum by increasing

cell proliferation; alternatively, may be

vestigial products of HERS with no function

in the mature tissues of the periodontium

No animal models with

defective ERM have

been developed

Hasegawa et al. (2003);

Yamashiro et al. (2003)

aNote: Amelogenin has several isoforms ( Bartlett et al ., 2006):

� LRAP (6.9 kDa); also called [A ‐ 4]/M59 and suggested to be a signaling molecule for odontoblasts and cementoblasts ( Tompkins and Veis, 2002 ). LRAP KO and

LRAP overexpression in mice do not appear to have a root phenotype.

� [Aþ 4]/M73 (8.1 kDa) has also been proposed as a signaling molecule ( Tompkins and Veis, 2002).

� TRAP: Similarly, suggested as a signaling molecule ( Swanson et al. , 2006).bStudies by Wang et al. (2005a) and Tompkins et al. (2006) have identified LAMPs as possible regulators of amelogenins, either involved in assisting with breakdown of

amelogenins (LAMP‐3, Wang et al.) or possibly serving as cell surface receptors (LRAP‐LAMP‐1, Tompkins et al.). Further, Tompkins et al. reported that A‐4/LRAP binds

to murine LAMP‐1, a lysosomal associated membrane protein, also present on cell surfaces, in a saturable fashion in murine myoblasts (C2C12 cells).

Hypothesized activitiesDifferentiationPrecursor cells

Origins of cementoblasts and cementum

Epithelial

Papilla

Follicle

IEE

OEE

Pulp/Odontoblast

HERS

Osteoblast

PDL Cell

Cementoblast

ERM

HERS dislocates from developing rootsurface and forms ERM (Cho and Garant,2000; Diekwisch, 2001; Luan et al., 2006)

HERS secretes acellular cementum (Bosshardt,2005; Zeichner-David et al., 2003)

HERS cells e-m transform and secretecellular cementum (Bosshardt, 2005; Bosshardtand Nanci, 2004; Lezot et al., 2000; Thomas,1995)

Follicle-derived cementoblastssecrete acellular and cellular

cementum (Cho and Garant, 2000;Diekwisch, 2001; Luan et al., 2006)

Follicle-derived cementoblastssecrete (only) cellular cementum(Chai et al., 2000; Zeichner-David et al., 2003)

HERS secretes proteins that inducecementogenesis (Fong and Hammarstrom,2000; Fukae et al., 2001; Gestrelius et al., 2000;Hu et al., 2001)

Generally established Proposed hypothesis

Induction of cementogenesis (Alatli-Kutet al., 1994; Takano et al., 2003)

Ectomesenchymal


as well as in root development, will require concentrated research eVorts in the

next decade.Knowledge built from these research findingswill enhance our basis

for regenerating the crown as well as the ‘‘whole’’ tooth.

III. Question 2. What Do We Know About the Cells Requiredfor Periodontal Development and Regeneration?

While the origins for cells and tissues of the tooth crown have been fairly

well established, much remains unclear about cells involved with forming the

periodontium, and this has been a subject of speculation for at least five

decades, arguably with no authoritative statement yet made. The following

section will discuss the cells involved in periodontal development, their

potential contribution to regeneration, as well as controversies regarding

their origins.

A. Developmental Cells

Odontogenesis is characterized by sequential, reciprocal, reiterative signaling

between tissues of the epithelium (dental lamina) and mesenchyme (ecto-

mesenchyme derived from cranial neural crest) and ultimately, both epithelial

and ectomesenchymal cells are involved in periodontal tissue formation

(Fig. 2). Tooth development continues with ectomesenchymal cells develop-

ing into the dental follicle and surrounding the epithelial enamel organ and

the mesenchymal dental papilla (Nanci and Somerman, 2003). Cells within

the follicle region have been proposed to be the origin for tissues of the

periodontium, namely cementum, periodontal ligament (PDL) and alveolar

bone. But this hypothesis has not been accepted without challenge, as will be

discussed in some detail below. The exact origin of cementum and cemento-

blasts remains a matter of debate; current hypotheses are summarized in

Fig. 2 and described in the following text.

Figure 2 Origins of cementoblasts and cementum. This figure reviews competing hypotheses on

origins of cementoblasts and cementum tissue by considering possible fates for cells of

ectomesenchymal (top panel) and epithelial (bottom panel) origin, and hypothesized roles

in tooth root formation. The primary division lies between a proposed ‘‘classical’’ mesenchymal

origin (represented in top panel) and an ‘‘alternative’’ epithelial origin (represented in bottom

panel). However, several variations exist within each hypothesis, and these diVerences need not be

mutually exclusive. IEE ¼ inner enamel epithelium; OEE ¼ outer enamel epithelium; HERS ¼Hertwig’s epithelial root sheath; ERM ¼ epithelial cell rests of Malassez; PDL ¼ periodontal

ligament; e‐m ¼ epithelial‐mesenchymal transformation.

64 Foster et al.

1. Ectomesenchymally Derived Cells

During the cap stage of tooth development, the epithelial enamel organ takes

on a concave form and is bordered by two ectomesenchymal tissues, papilla

and follicle, descended from cranial neural crest (CNC) cells. The dental

papilla is composed of densely packed cells that during the subsequent bell

stage become increasingly sequestered within the developing enamel organ,

eventually giving rise to the pulp and dentin tissue. The mesenchymal cells

surrounding the developing enamel organ and papilla compose the dental

follicle (sometimes called the dental sac), a collagenous tissue separating the

nascent tooth bud from surrounding oral tissues. Dental follicle has been

proposed to be the common origin for supportive tissues of the tooth (i.e.,

the periodontium), including cementum, PDL, and alveolar bone (Cho and

Garant, 2000; Nanci and Somerman, 2003; Saygin et al., 2000). Cells within

the follicle region are also essential for signaling associated with tooth

eruption, through regulation of osteoclasts in the coronal portion of the

bony crypt via CSF‐1, RANKL, and OPG expression, signaled in turn by

PTHrP and other factors still to be identified (Liu et al., 2005a; Wise et al.,

2002, 2005). During tooth eruption and root elongation, the formative

dental follicle gives rise to the mature structure of the PDL, a highly vascular

and innervated region that provides attachment of the tooth to the sur-

rounding alveolar bone via collagen fibers. The PDL is also home to a

heterogeneous population of cells, including stem cells with potential for

regeneration of periodontal tissues, which will be discussed in more detail at

the end of this section (Cho and Garant, 2000; Nanci and Somerman, 2003;

Seo et al., 2004).

2. Hertwig’s Epithelial Root Sheath Cells

Root initiation begins after the crown dentin and enamel have formed, and

before tooth eruption. The cervical loop, the most apical extension of the

enamel organ, extends into the bilayered Hertwig’s epithelial root sheath

(HERS), composed of the outer enamel epithelium (OEE) and inner enamel

epithelium (IEE). The HERS layers proliferate and extend apically, outlining

the future shape of the nascent tooth root (Luan et al., 2006). In mammalian

root formation, dislocation from the root and disintegration of the double‐layered HERS is considered a key event, allowing access of the underlying

dentinal surface to cementum‐forming cells (Cho and Garant, 1988;

Diekwisch, 2001). This general sequence of events has been further supported

by in vitro tissue recombination experiments (MacNeil and Thomas, 1993). As

root formation continues, the dislocated HERS cells break up into epithelial

‘‘nests’’ and ‘‘cords,’’ which may be subsequently reduced to epithelial cells

rests of Malassez (ERM) (Wentz et al., 1950). In addition to the possibility


of HERS cells migrating away from the root surface to contribute to ERM,

it has also been documented that some HERS cells undergo apoptosis

(Kaneko et al., 1999) or become incorporated into the cellular cementum

(Lezot et al., 2000). Developmental studies, as well as a review of evolution-

ary evidence (Luan et al., 2006), provide information indicative of a role

for ERM in regulating PDL homeostasis, protecting against resorption

and ankylosis, and perhaps contributing to cementum repair (Hasegawa

et al., 2003).

It has been proposed that HERS plays an active role in induction

or secretion of acellular and/or cellular cementum, and this hypothesis is

described in detail below (under the ‘‘alternative’’ epithelial hypothesis of

cementogenesis). Potential signals of HERS that may stimulate cementum

formation are discussed under Section III and listed in Table II.

B. Derivation of Cementum: Competing Theories of Cementoblast Origin

1. Acellular Versus Cellular Cementum

Acellular cementum covers approximately two‐thirds of the root, and

around the time the tooth comes into occlusion, cementum development

shifts from acellular to cellular. Acellular cementum (acellular extrinsic fiber

cementum, AEFC) forms first on the coronal and mid‐portion of the root at

a slow rate, while cellular cementum (cellular intrinsic fiber cementum,

CIFC), a more bone‐like tissue, forms more apically and more rapidly,

incorporating cells into the mineralized matrix that become cementocytes.

Acellular cementum seems to be more dependent on alkaline phosphatase

activity (Jayawardena et al., 2002), as it may be more severely aVected than

cellular cementum in hypophosphatasia (Beertsen et al., 1999; van den Bos

et al., 2005).

The cause or mechanism of the shift from acellular to cellular cementum is

not well understood, though hypotheses to explain this transition include the

possibilities that occlusal mechanical forces somehow cue the shift, cells

producing each type of cementum are from diVerent populations, or diVer-ent extracellular factor(s) regulate(s) acellular versus cellular cementum.

Potential regulators that have been considered include the dentin matrix of

the root, enamel matrix proteins, and other components of the ECM.

In experiments modeled after those performed by Hammarstrom (Alatli‐Kut et al., 1994; Hammarstrom et al., 1996), Takano et al. treated rats and

guinea pigs with bisphosphonate to delay dentin matrix mineralization, and

observed that acellular cementum was precluded by formation of a cellular

type of cementum on the nonmineralized dentin along the entire surface of

the root (Takano et al., 2003). While dentin sialoprotein (DSP) was localized

66 Foster et al.

to the border between dentin and cellular cementum (but not acellular

cementum) in untreated rats, in the bisphosphonate‐treated rats DSP pene-

trated the soft dentin matrix along much of the root, even diVusing into

surrounding tissues. The authors hypothesize that the timing of mineraliza-

tion of mantle dentin in conjunction with dentin matrix proteins influences

the type of cementum forms.

2. The ‘‘Classical’’ Mesenchymal Hypothesis

The ‘‘classical’’ hypothesis, nearly 50 years old (Paynter and Pudy, 1958),

proposes cementoblasts are cells descended from the dental follicle that

migrate to the developing root surface and are triggered to diVerentiate intocementum matrix‐secreting cells, that is, cementoblasts (Bosshardt and

Selvig, 1997; Cho and Garant, 2000; Diekwisch, 2001; Luan et al., 2006;

Saygin et al., 2000). This hypothesis fits into an overarching proposition of a

common developmental origin (i.e., the dental follicle) for the three forma-

tive cell populations of the periodontium, namely cementoblasts, PDL cells,

and alveolar osteoblasts (Melcher, 1985; Ten Cate, 1997).

During rat molar root development, mesenchymal cells of the follicle were

reported tomigrate to theHERS, disrupt the epithelial structure, and begin to

lay down cementum matrix via cellular processes, as interpreted from studies

employing light and electron microscopy (Cho and Garant, 1988, 2000).

Similar observations of mesenchymal cells accessing the developing root

surface were reported in the mouse molar, with the exception that HERS cells

may themselves contribute to the disruption of the HERS structure prior to

root formation, while the first matrix secreting cells in cementum formation

were the migrating mesenchymal (follicle) cells (Diekwisch, 2001; Luan et al.,

2006; Ten Cate, 1997). Migratory capabilities of dental follicle cells were

supported in mouse molar organ culture, where fluorescently tagged follicle

cells migrated apically and were found in PDL and alveolar bone (Diekwisch,

2002). Human and porcine specimens in the extensive Bernard Gottlieb

collection (Baylor College of Dentistry, Dallas, TX) yielded similar observa-

tions that HERS cells departed the root surface prior to initiation of cemen-

tum, forming a loose network of cells that subsequently disintegrated, with

some presumably contributing to the population of ERM, islands of

epithelial‐derived cells that remain in the PDL into adulthood with uncertain

function (Diekwisch, 2001).

In support of the ‘‘classical’’ hypothesis, in a well‐executed developmental

study in mice, Chai et al.were able to track cells of cranial neural crest (CNC)

origin, from embryogenesis to 6 weeks of age, using a two‐component

(Wnt1‐Cre, R26R) genetic system for cell lineage tracking through develop-

ment (Chai et al., 2000). In this way, CNC progeny were identified by

�‐galactosidase activity (only present in cells expressing Wnt1 and constitutive


R26R, but marked indelibly, even when Wnt1 expression is shut oV). CNC‐derived cells contributed to formation of cementum and periodontal

ligament, as well as to condensed dental mesenchyme, dental papilla, odonto-

blasts, and other tissues. While cementum showed strong lacZ expression,

indicating a CNC origin, these results need not preclude an epithelial

contribution.

Some species‐specific diVerences in cementum development are worth

noting, one being that in rodents the sequence of events is muddied by

HERS initially covering the entire root surface and remaining in close

proximity as cementum is formed, as opposed to humans where HERS is

more completely divorced from the developing root prior to any observable

cementum.

Supposing the classical hypothesis of common origin for cellular and acellu-

lar cementum, PDL, and alveolar bone, the question naturally arises, ‘‘What

factors direct a common precursor cell to become a cementoblast, osteoblast,

or PDL cell?’’ This is a valid question worthy of future study, with some

potential regulators discussed under Question 3 and presented in Tables II–V.

3. The ‘‘Alternative’’ Epithelial Hypothesis

An alternative hypothesis that has been proposed (Slavkin and Boyde, 1975)

questions the evidence for a mesenchymal origin and instead considers an

epithelial contribution from HERS to cementogenesis (Bosshardt, 2005;

Bosshardt and Nanci, 1997, 2004; Bosshardt and Schroeder, 1996; MacNeil

and Somerman, 1999; Thomas, 1995; Zeichner‐David, 2006; Zeichner‐David

et al., 2003). Under this proposal, cementoblasts are thought to be derived

from an epithelial‐mesenchymal transformation of HERS cells, which then

secrete cementum matrix proteins. DiVerences of opinion exist regarding

origins of acellular and cellular cementum, as delineated below.

In a careful observation of cementogenesis in pigs using light microscopy

and TEM with immunogold labeling, Bosshardt and Nanci found a lack of

compelling evidence for a mesenchymal migration of follicle cells, but rather

observed a potential phenotypic epithelial‐mesenchymal transformation of

outer enamel epithelium (OEE) cells to a secretory, connective tissue cell‐likemorphology in the vicinity of initiation of cementogenesis (Bosshardt and

Nanci, 2004). Studies using a Dlx‐2/LacZ reporter construct in transgenic

mice localized Dlx‐2 expression to root epithelium (HERS) during root

development, and also to a limited population of cementoblasts during

acellular and cellular cementum formation, but failed to detect Dlx‐2 in

dental follicle and papilla (Lezot et al., 2000). During acellular cementum

formation, Dlx‐2 was identified in diVerentiated cementoblasts, and during

cellular cementum formation in innermost cementoblasts and cementocytes.

68 Foster et al.

Some of the Dlx‐2 positive cementoblasts also stained positive for amelo-

blastin. The authors concluded a complex origin for cementum‐forming

cells, in other words, suggesting that a select population of cementoblasts

were derived from the HERS. Another interpretation of these results could

be that HERS cells are passively incorporated within the forming cementum

matrix being synthesized by mesenchymally derived cementoblasts.

Evidence for an epithelial origin for acellular cementum also lies in the

demonstration that these cells can produce proteins characteristic of mesen-

chymal cells, and cementum in particular (Bosshardt and Nanci, 1997;

Mouri et al., 2003; Zeichner‐David, 2006; Zeichner‐David et al., 2003).

If HERS cells transform to contribute to acellular cementum formation,

the possibility of cellular cementum derived from HERS may also be con-

sidered. A hypothesis based on morphological examinations in human and

porcine teeth proposes that HERS is the origin not only for both types of

cementum, but also for subpopulations of periodontal ligament fibroblasts

(Bosshardt, 2005). This hypothesis would explain the diVerent phenotypes ofcementoblast versus osteoblast, and the heterogeneity of cells populating the

PDL region. While strides are being made toward describing the origins of

cementum, and a great dialogue of diVering viewpoints has been cultivated

in the literature, the origin of cementum is still under debate.

4. Involvement of Epithelial‐Derived Products in Cementum Formation

Apart from ideas about the transformation of HERS to cementoblasts, it has

been suggested that HERS may induce cementogenesis by secretion of enamel

matrix proteins (EMPs) (e.g., amelogenin, ameloblastin, and enamelin) or

other proteins that influence cell migration, attachment, and/or matrix secre-

tion leading to cementogenesis (Gestrelius et al., 2000; Hammarstrom et al.,

1996; Slavkin, 1976; Zeichner‐David, 2001, 2006).

Evidence supporting a role for EMPs in cementogenesis has been ac-

cumulating from investigations employing immunohistochemistry, in situ

hybridization, and in vitro assays, all supporting EMP expression by HERS

cells in several species (Bosshardt and Nanci, 1998; Fong and Hammarstrom,

2000; Fukae et al., 2001; Hamamoto et al., 1996; Hammarstrom, 1997; Hu

et al., 2001; Luo et al., 1991; Slavkin et al., 1989a,b). However, serious dis-

crepancies in these collective reports remain unresolved. Reports conflict with

one another on several points, including: which EMPs are or are not ex-

pressed, how much protein is present and if levels are suYcient to play an

important role in root formation, the region of localization on the root, and

what cells produce EMPs. In studies of porcine cementogenesis, little evidence

was found to support a significant role of enamel matrix derivatives (in this

case, amelogenin and ameloblastin) based on absence of significant quantities

of these ameloblast products in the HERS and on the developing root surface


(Bosshardt and Nanci, 2004). In mouse molars, immunocytochemistry and

in situ hybridization failed to detect any trace of amelogenin in HERS cells,

and amelogenin was also absent in porcine cementum extracts assayed by

Western blot (Diekwisch, 2001). Janones et al. used microwave processing in

conjunction with immunocytochemistry to demonstrate that in developing

rat molars, amelogenin was present in early tooth formation, but gone before

initiation of cementogenesis (Janones et al., 2005). These conflicting results,

both old and new, should then be considered carefully for possibilities such as

false positives and specificity or cross‐reactivity of antibodies. Ultimately, to

confirm that EMPs are functionally important in cementogenesis, a consistent

and regular expression of these proteins would be expected in association with

developing cementum, and up to now, this standard remains to be met in a

convincing way. Better probes, antibodies, etc., should assist in solving this

puzzle.

For example, an immortalized murine HERS cell line expressed amelo-

blastin (but not amelogenin or enamelin) in vitro. HERS conditioned media

was found to induce BSP and OCN expression, as well as in vitro minerali-

zation (Zeichner‐David, 2006). Further, theseHERScellswere also observed to

undergo an apparent phenotypic transformation to a morphologically distinct

fibroblastic cell expressing cementum‐associated transcripts BSP, OCN, and

OPN, supporting the potential not only for HERS to induce cementogenesis,

but also secrete cementum matrix proteins directly (Zeichner‐David et al.,

2003).

Furthermore, if EMPs play an important role in tooth root formation,

a cementum phenotype might be expected in animals deficient in these

proteins. While a root phenotype has been suggested in amelogenin knock-

out mice, it is unclear whether this is a direct or indirect result (Hatakeyama

et al., 2003, 2006), and this will be addressed in the next section under

Question 3, as well as in Table II focusing on HERS‐ and ERM‐associatedproducts. Though the role of epithelial proteins in root formation remains

controversial, a treatment derived from porcine enamel organ known as

EmdogainÒ is currently used clinically with the aim to promote periodontal

regeneration. The applications of EmdogainÒ will be discussed in more

detail under Question 3.

C. Differences Between Cementoblasts and Osteoblasts

A common origin for cementum and alveolar bone has been proposed in the

form of the dental follicle and perifollicular cells. Yet in the absence of a

clear understanding of cementum origins, how can progress be made toward

improving tissue engineering and promoting periodontal regeneration?

Cementoblasts and osteoblasts and their respective tissues may be compared

70 Foster et al.

with respect to cells and regulators of their diVerentiation, and structural

and functional properties of the cementum versus bone matrices. It is outside

the scope of this review to exhaustively catalog points of comparison be-

tween cementoblasts and cementum versus osteoblasts and bone; for this the

reader is recommended to excellent reviews on the topic (Bosshardt, 2005;

Diekwisch, 2001; Nanci and Bosshardt, 2006; Saygin et al., 2000; Zeichner‐David, 2006). Potential areas for progress in characterizing cementoblasts

including identification of cementum marker proteins, performing compar-

isons to other cell types, and using in vitro models of cementoblasts and

precursor cells in conjunction with in vivo observations.

1. Cementum‐Specific Markers

Attempts have been made to identify unique cementum‐specific marker pro-

teins that would distinguish cementum from bone. In the study of dental

tissues, many ‘‘specific markers’’ have even been declared, later to be reported

in other tissues as well. For example, DSPP and DMP‐1, formerly thought

dentin‐specific, have subsequently been localized to bone and cementum and

their respective cells, in vivo and in vitro (Baba et al., 2004a; Foster et al., 2006;

Qin et al., 2002). Amelogenin, thought to be an ameloblast‐specific product, isexpressed by pulp cells and odontoblasts during tooth development (Nagano

et al., 2003; Oida et al., 2002; Papagerakis et al., 2003; Veis et al., 2000).

There is a history of putative cementum‐specific factors aswell. A cementum‐derived growth factor (CGF) originally isolated from a human cementoblas-

toma and posited to be a novel growth factor and mitogen (Yonemura et al.,

1992, 1993) was identified in human and bovine cementum, as well as in PDL

cells and furthermore, on detailed analysis recognized as being very similar in

composition to IGF1 (Narayanan et al., 1995). Cementum attachment protein

(CAP) was identified from a human cementum tumor and proposed to be an

extracellular matrix protein functioning in migration and attachment of ce-

mentoblast precursors to the root surface (Arzate et al., 1992; Bar‐Kana et al.,

2000; Pitaru et al., 1995, 2002; Saito et al., 2001); CAP was later found to be

expressed in PDL cells and alveolar bone cells, and to share homology with

some collagen domains (BarKana et al., 1998;Wu et al., 1996). Another protein

identified from cementum tumor was termed cementum‐protein 23 (CP‐23)(Alvarez‐Perez et al., 2006). Antibodies made to this protein cross‐reacted with

a cartilage type collagen, type X collagen, and CP‐23 was identified within the

PDL region, cementum and around blood vessels in the PDL. While CGF,

CAP, and CP‐23 may play roles in periodontal development, they are not,

strictly speaking, markers of cementoblasts or cementum. Importantly, these

proteins were identified from a human cementoma and cementomas by defi-

nition are composed of a variety of cells, for example, fibroblasts, osteoblasts,

and cementoblasts. Additional examples include lumican and fibromodulin,


reported to be more highly expressed in cementum than bone (Bosshardt,

2005). Glucose transporter‐1 (GLUT‐1) was suggested to be a factor separat-

ing cementoblasts from osteoblasts (Koike et al., 2005), and though this

protein is widely expressed, it is tenfold higher in human cementoblasts versus

osteoblasts in vitro.

While these proteins may not be unique cementum markers, they may still

be useful in defining cementum matrix versus bone. These and other proteins

are thought to be enriched or relatively highly expressed in cementum versus

bone and have potential to be used to assemble a panel of markers charac-

teristic or suggestive of cementum. As of yet, there is not any marker by itself

that is unique or specific to this tissue.

2. Comparisons of Cementoblasts to Other Cell Types

As no conclusive study demonstrating cementoblast origin has yet been

reported and no cementum‐specific marker is likely, a very practical option

may be direct cell‐to‐cell comparison, as between cementoblasts and osteo-

blasts. In vivo studies are limited by the need for specific probes and anti-

bodies and the laborious nature of screening, while in vitro studies make

many aspects of analysis easier, but results must be analyzed cautiously

because of removal of cells from the natural milieu. Head‐to‐head compar-

isons of cells have yielded valuable insights when confirmed by other meth-

ods such as in situ hybridization and immunohistochemistry. Examples of

such comparison technologies include laser capture, microarray analysis,

proteomics, and subtractive hybridization. All, except laser capture, have

been used to begin to define markers for dental cells (Hao et al., 2005; Koike

et al., 2005; Lallier et al., 2005; Reichenberg et al., 2005; Shi et al., 2001).

Care must be taken in the choice and preparation of cells to be compared in

such experiments, as this sort of analysis may result in misleading conclu-

sions if precautions are not used. For example, the cell populations being

compared may be derived from diVerent developmental stages, which would

strongly influence gene and protein profiles expressed.

The logical comparison for cementoblasts would be osteoblasts lining the

surrounding alveolar bone. Although alveolar bone is generally thought to be

consistent with other bone tissues in cell and matrix components, it is a local,

specialized bone tissue with unique features, including proximity to the tooth

and the cellular/molecular influence of the tooth tissues, and a very high rate

of remodeling relative to other bone tissues of the body (Sodek and McKee,

2000). There is some evidence that bonemarrow stromal cells (BMSCs) within

the same individuals diVer in a skeletal site‐specific fashion, and that orofacialstem cells may represent a unique population (Akintoye et al., 2006).

If cementoblasts and alveolar osteoblasts share a direct precursor cell, it is

72 Foster et al.

possible that they share a more similar genetic profile than cementoblasts

versus other osteoblast or osteoblast precursor populations.

A cleverly designed experiment by Kaneda et al. used a strategy of

consecutive enzymatic digestions of extracted mouse molars to explore

diVerences between subpopulations of PDL cells, from cells obtained mid-

way across the PDL space to those closest to the root surface, including

cementum‐lining cells (Kaneda et al., 2006). As subpopulations were char-

acterized closer to the root, their alkaline phosphatase activity and potential

for promoting in vitro mineralization increased, as well as expression of BSP

mRNA. Further studies employing a similar approach should yield insights

into characteristics of subpopulations of cells located in the PDL region,

and into the potential of these various subtypes to diVerentiate toward a

cementoblast phenotype.

3. In Vitro Cell Models for Cementoblasts and Precursor Cells

Establishment of in vitro cementoblast models in parallel with studying

in vivo cementum development can be a powerful way to progress our

understanding of the origins and characteristics of this tissue. To date,

cementoblast cell lines for use in vitro have been prepared from mice (Berry

et al., 2003; D’Errico et al., 2000; MacNeil et al., 1998), rats (Kitagawa et al.,

2005), cows (Saito et al., 2005), human (Grzesik et al., 1998), and human

cementoblastoma (Arzate et al., 1992). These cells express high levels of BSP,

OCN, and OPN, and can produce mineralized nodules in vitro and ectopic

ossification in an in vitro SCID mouse model. Additionally, putative cemen-

toblast precursors, dental follicle cells, have been isolated and cultured from

mice (Zhao et al., 2001), rats (Yao et al., 2004), and humans (Morsczeck

et al., 2005), and these may provide clues as to potential mechanisms

required cementoblast diVerentiation. An immortalized HERS‐derived cell

line has been established from mice, and has been characterized as producing

enamel‐related proteins prior to a phenotypic shift toward a mesenchymal

cell type that produces a mineralized matrix resembling acellular cementum

(Zeichner‐David et al., 2003). While species diVerences, phenotypic drift,

and secondary eVects of immortalizationmust be considered, these approaches

have already yielded considerable insight into the nature of ‘‘cementoblasts’’

and will continue to do so in future research.

D. Tooth Stem Cell Populations

The nature and regenerative capacities of stem cell populations in tooth

tissues have been one of the most exciting revelations in dental research

in the last five years, with enormous potential for future application in


designing regenerative therapies and tooth engineering in the future (Bartold

et al., 2006a; Chai and Slavkin, 2003; Fong et al., 2005; Ohazama et al.,

2004b; Risbud and Shapiro, 2005; ThesleV and Tummers, 2003). Embryonic

stem cells are pluripotent, that is, they have the capability to diVerentiateinto all cell types with appropriate conditions and stimulation. Stem cell

research eVorts focus on the sizeable probability for such cells to be used in

adult tissue regeneration and gene therapy. However, the current number of

embryonic stem cell lines is limited and their use is controversial and subject

to government regulation. As a result, there has been great interest in

exploring stem cell populations in adults. Adult stem cells are undiVeren-tiated cells that remain in developed tissues of the adult organism and are

multipotent, meaning they have the capability to diVerentiate into multiple

cell types within a tissue, organ, or system. Adult stem cells have been

identified in several locations including bone marrow, blood, neural and

muscle tissue, and tooth environment (Fuchs and Segre, 2000). While the

breadth of potential for diVerentiation, or potency, for most of these adult

stem cell types remains to be fully explored, the therapeutic possibilities for

an adult‐derived, unlimited population of multipotent stem cells are quite

exciting (Robey, 2000). The identification and characterization of these adult

stem cell populations in the tooth region has been one of the most exciting

and promising discoveries of the last five years.

1. Dental Pulp Stem Cells

The dental pulp holds promise for regeneration of dentin in response to

trauma (Goldberg and Smith, 2004), and this has been recognized for many

years. This knowledge, coupled with advances in technology, has enhanced

our understanding of the underlying mechanisms involved in pulp cell

maturation. A human adult stem cell population was identified and isolated

from pulp chambers of impacted third molars. In cell culture, these dental

pulp stem cells (DPSCs) were demonstrated to be clonogenic, rapidly pro-

liferative, able to diVerentiate and form mineralized nodules in vitro, and

produce a structure resembling a dentin/pulp complex in ex vivo transplan-

tation experiments with SCID mice (Gronthos et al., 2000). In the same

experiment, bone marrow stromal stem cells (BMSSCs) formed a more

distinctly bone‐like tissue. In subsequent studies, the DPSC profile was

further developed by identifying mesenchymal stem cell markers STRO‐1and CD146, and transplant experiments were performed with DPSCs, with

cells exhibiting odontoblast‐like gene and protein expression, and produc-

ing dentin‐like tissues (Batouli et al., 2003; Shi et al., 2001). The DPSC niche

was hypothesized to be a perivascular location within the pulp. Subsequent

work demonstrated the ability to harvest similar mesenchymal stem cells

from human exfoliated deciduous teeth, cells that were termed SHED

74 Foster et al.

(Miura et al., 2003). Sorting and ex vivo expansion of pulp stem cells from

exfoliated deciduous teeth allowed for the cells to be directed to adipocyte

and myotube phenotypes, as well as osteoblast‐like cells that produced a

mineralized tissue consistent with woven bone (Laino et al., 2006).

2. Periodontal Ligament Stem Cells

The PDL demonstrates some limited potential for repair of periodontal

tissues should they be damaged by trauma or disease, however, while there

are currently several strategies aimed at regenerating periodontal tissues,

sometimes successful, they are not predictable (Grzesik and Narayanan,

2002; Taba et al., 2005; Wang et al., 2005b; Zohar and Tenenbaum, 2005).

This repair potential of the PDL is thought to result from the presence of

a population of multipotent stem cells within the local region or recruited

from the vasculature that are capable of regenerating cementum, bone, and

PDL fibers (Bartold et al., 2000; Gould et al., 1980; McCulloch, 1985, 1995;

Melcher, 1985). Although several groups have demonstrated the regene-

rative potential of a compartment of periodontal cells, recent studies

have confirmed a stem cell population and characterized the nature of these

cells.

Human postnatal PDL stem cells (PDLSCs) were isolated, cultured, and

characterized in vitro (Seo et al., 2004). PDLSCs were fibroblast‐like, clono-genic and rapidly proliferative, and were positive for mesenchymal stem cell

markers STRO‐1 and CD146, similar to DPSCs and BMSSCs, indicating a

possible common perivascular origin. Interestingly, expanded PDLSCs also

expressed relatively high levels of a tendon‐associated transcription factor,

scleraxis. In vitro studies showed that after incubation in diVerentiationmedia, PDLSCs expressed proteins characteristic of cementoblasts, including

BSP, OCN, MEPE, ALP, and TGF�R1, and had the ability to promote the

formation of mineralized nodules. PDLSCs transplanted into SCID mice

produced collagen fibers suggestive of the PDL and amineralized tissue consis-

tent with cellular cementum. It remains unclear what signals may be necessary

to cue precursor cells to a cementum versus bone phenotype, and at present, no

specificmarkers have been established for cementum versus bone (as described

in detail above). Subsequent studies added to this work by showing that

viable PDLSCs could be retrieved from frozen PDL tissues (Seo et al., 2005),

increasing the practical potential for these stem cells to be used clinically

(Bartold et al., 2006a; Shi et al., 2005). While the origins of cementoblasts

remain in question, studies with periodontal ligament stem cells (PDLSCs)

showing ability to produce cementum‐like tissues in SCID mouse transplant

experiments lend some support to the mesenchymal origin of cementum, or

at least cellular cementum. Indeed, expression of common transcription

factors, cell surface markers, growth factors, and matrix proteins in postnatal


stem cell populations suggests common regulatory pathways for cementum,

dentin, and bone.

3. Epithelial Stem Cells of the Continuously Erupting Incisor

The postnatal stem cells of the bone marrow, dental pulp, and PDL are

multipotent mesenchymal stem cells with a capacity for limited generation of

mesenchymally derived tissues. In the teeth of humans and the molars of

rodents (teeth of limited eruption), the ameloblasts that form the tooth

enamel are lost on tooth eruption, and there seems to be no epithelial self‐renewing stem cell population remaining. However, in the continuously

erupting incisor of rodents, new enamel (as well as dentin and cementum)

are constantly generated apically to compensate for attrition on the incisal

edge (Harada et al., 2002a). Therefore, new ameloblasts must be available

from postnatal epithelial stem cell populations in order to synthesize enamel

in the adult. A specialized apical bud structure proposed to be the epithelial

stem cell niche was identified at the apical end of incisors of mice and guinea

pigs (Ohshima et al., 2005). The apical bud was characterized by large

amounts of stellate reticulum and basal epithelium, and a candidate mole-

cule for maintenance of the apical bud niche was identified as fibroblast

growth factor 10 (FGF‐10) by adjacent mesenchymal cells, with epithelial

Notch signaling also implicated as playing a role (Harada et al., 2002b).

Ameloblast diVerentiation and patterning in the rodent incisor was shown

to be dependent on downregulation of follistatin in the epithelium on the

labial edge, and subject to regulation by the antagonistic actions of BMP‐4fromodontoblasts and activin fromdental follicle (Yamashiro et al., 2004). In a

primary cell culture study, apical bud stem cells were shown to require mes-

enchymal cell interaction to be prompted to diVerentiate to an ameloblast‐likephenotype (Morotomi et al., 2005).

4. Crown and Root Developmental Fates

The developmental diVerence between teeth of limited eruption versus con-

tinuous eruption may lie in the ‘‘choice’’ between maintenance of the epithe-

lial stem cell niche as opposed to loss of this niche and development of a

root. The developmental fate of the continuously erupting incisor is deter-

mined by maintenance of an apical bud epithelial stem cell population

(Ohshima et al., 2005), while if crown development is arrested, a root fate

is pursued (Tummers and ThesleV, 2003). The root fate is characterized by

transformation and flattening of the stellate reticulum into the double‐layeredHERS,which lengthens to define the root shape outline, and fenestrates

just prior to cementum formation. The fate of the HERS is a matter of debate,

potentially becoming ERMs, secreting pro‐cementum factors, or contributing

76 Foster et al.

to acellular and/or cellular cementum formation. Crown and root fates were

investigated in an elegant study of the molar of the sibling vole, which intrigu-

ingly develops root analog areas while remaining continuously erupting. In the

root‐like regions of vole molars, FGF‐10, Notch 1 and 2, and BMP‐4 signals,

thought to contribute to the epithelial stem niche, disappear coincident with

development of root‐like tissues, similar to in mouse molars (Harada et al.,

2002b; Ohshima et al., 2005; Tummers and ThesleV, 2003; Yokohama‐Tamaki

et al., 2006). Therefore, mechanisms that downregulate signals specific for

crown formation may be required for initiation of root formation. These

findings are intriguing to consider in light of the failure of tissue recombination

experiments to form tooth roots.

IV. Question 3. What Genes and Associated Proteins AreImportant for Root/Periodontal Tissue Formation?

In the last 5 years, newly discovered factors and new roles for already known

factors regulating root/periodontal development have emerged and have

changed how we view odontogenesis. Much of the recent progress in under-

standing factors that regulate root/periodontal tissue (R/PT) development

has naturally arisen from studies of molecules that control mineralized tissue

formation. The development of transgenic mice, designed to over‐ or under-express specific genes, and the study of mice with well‐defined mutations has

provided much insight into potential factors required for R/PT development.

Tables II–V highlight factors that have been reported within (approximately)

the last 5 years to play a role in R/PT development, including genes/

proteins that result specifically in a root phenotype when deficient or over-

expressed. In addition, factors already established to play a role in root/

periodontal development will be briefly discussed, with an update of relevant

references.

It is well established that specific genes and associated proteins are re-

quired for patterning, proliferation, and diVerentiation of cells during crown

development, that is ameloblasts for enamel and odontoblasts for dentin.

Much attention has been given to factors involved in epithelial–mesenchymal

interactions, including fibroblast growth factors (FGFs), sonic hedgehog

(SHH), bone morphogenetic proteins (BMP)s, Wnts and associated recep-

tors, as well as downstream transcription factors such as distal‐less homeo-

box (Dlx), Msx, AP‐1 factors, Pax‐9, and runt‐related transcription factor 2

(RUNX 2). Many of the knockout (KO) models developed to understand the

specific roles for these genes during tooth development have resulted in

severe phenotypes (and sometimes death in utero) because of the critical role

these genes/proteins play during early development. This has in some cases


prevented any detailed analysis of the teeth, because if the animals survived,

tooth development was arrested at stages prior to root formation. Conse-

quently, there is limited direct evidence for the role of such genes and their

products in R/PT formation (Zhao, 2003). Several excellent reviews on the

roles of specific genes and signaling molecules during crown development are

available (Jernvall and ThesleV, 2000; ThesleV and Aberg, 1999; ThesleV and

Mikko la, 2002; Thes leV et al. , 1995 , 2 003; Tucker and Sha rpe, 2004; Zhanget al., 2005). One major finding from research targeting crown development

has been the recognition of niche areas, specifically the enamel knot, and

cervical loop/apical bud regions, where a plethora of genes crucial for reg-

ulating crown development are expressed.

While the aforementioned studies have focused primarily on molecules

regulating crown development, they have also raised new questions related

to investigation into R/PT development. Specifically, do ‘‘niche’’ regions for

R/PT development exist, similar to the enamel knot region? And can we

identify the signaling molecules that regulate cell diVerentiation toward a

cementoblast, PDL cell, and osteoblast cell fate? Discussed below and

described in Tables II–V are factors, both well‐established and putative, that

have been implicated in regulating R/PT development, with an emphasis on

cementum formation. Some molecules that have been reviewed previously

(Bosshardt, 2005; Bosshardt and Nanci, 1997; Diekwisch, 2001; Popowics

et al., 2005; Saygin et al., 2000) or implicated in R/PT formation with limited

evidence to support their function in root development are mentioned below,

but not included in the tables.

A. Factors Associated with the Putative Epithelial Niche (HERS and ERM)and Surrounding Mesenchyme

After crown formation is completed, but prior to eruption, the outer and

inner epithelia form a double‐layered sheath called Hertwig’s epithelial root

sheath (HERS), and proliferate apically to outline the form the root will

take, as detailed in the previous section. It is generally accepted that HERS

cells of the inner enamel epithelium (IEE) regulate cells of the dental papilla

to diVerentiate into odontoblasts and secrete matrix proteins required for

forming root dentin, yet the events and molecular factors directly responsi-

ble for this sequence of events remain unknown (Thomas, 1995). With

further studies, specific factors critical for directing root dentin formation

may be identified (Table II).

The role of HERS with regard to cementum formation is even less clear,

and hypotheses are discussed in the previous section and summarized in

Fig. 2. Hypotheses discussed above include the induction of cementogenesis

by epithelial products from HERS, epithelial–mesenchymal transformation

78 Foster et al.

of HERS cells into a cementoblasts, and potential for diVerent contributionsin the development of acellular versus cellular cementum. After development

of the R/PT, remnant epithelial cells may reside in mature PDL as epithelial

cell rests of Malassez (ERM).

Several groups have begun to characterize HERS cells and their proteins

to determine the contribution of these cells/factors during tooth root devel-

opment. These investigations have demonstrated unique signaling molecules

within the HERS region, with preliminary evidence of a unique niche region.

A specialized structure termed the ‘‘apical bud’’ in the continuously erupting

rodent incisor has been identified as an attractive candidate for consider-

ation as an epithelial stem cell niche region for the crown (labial) side, with a

less well‐defined region on lingual side, that still may serve as a niche region

(Harada and Ohshima, 2004). In order to confirm that the HERS and labial

epithelial regions are stem cell niche regions, specific gene signals and target

cells need to be further characterized, and the possibility for epithelial‐mesenchymal transformation in some select group of cells considered. Some

of the investigations to date that support a role for HERS/epithelial pro-

ducts in R/PT formation are discussed below and also presented in Table II.

Signaling between cervical dental tissues directs the behavior of cells and

decision to act toward formation of crown versus root tissues. In the case of

continuous crown formation, dental papilla cells surrounding the cervical

loop express BMP‐4 and FGF‐10 (Table II). By regulating cell division,

FGF‐10 promotes survival of epithelial stem cells within the cervical loop

and allows continuous growth of rodent incisors. The data to date provide

evidence that the absence of these signals from the cervical region of mouse

molars, teeth of limited eruption, switches cellular activities from crown

formation to root formation (Tummers and ThesleV, 2003; Yokohama‐Tamaki et al., 2006).

Signaling between HERS cells and adjacent mesenchyme occurs during

root formation and appears to regulate the proliferation and diVerentiationof both epithelial and mesenchymal cells. Because the BMP–Msx pathway is

known to elicit reciprocal interactions between epithelial and mesenchymal

cells during early tooth development, Yamashiro and colleagues examined

the expression of BMP–Msx signaling pathway molecules within the HERS

region (Yamashiro et al., 2003). During tooth morphogenesis, expre-

ssion of BMP‐4 in the apical mesenchyme precedes Msx2 expression in the

root sheath. None of the BMPs (i.e., BMP‐2, 3, 4, or 7) were detected in the root

sheath epithelium, nor transcripts for Msx1 or 2 in the mesenchyme. In con-

trast, Yamamoto and colleagues found BMP‐2 and 4 expression in HERS

cells (Yamamoto et al., 2004a), suggesting that these signaling molecules

may play a role in developing root shape. In relation to cell diVerentiation,BMP‐2 and 7 are transiently expressed in both preodontoblasts and diVer-entiating odontoblasts, and may signal epithelial diVerentiation and/or have


autocrine or paracrine eVects on diVerentiating odontoblasts (Yamashiro et al.,

2003). Msx2 null mice have been reported to have irregularly shaped molar

roots, although the extent of these alterations is not clear (Satokata et al., 2000).

The expression of Msx2 in cells within the HERS region and of BMPs in

the surrounding mesenchyme/follicle cells, coupled with the root pheno-

type in Msx2 KO mice, suggests that these molecules play a role in regulating

root patterning and cell diVerentiation similar to their function during crown

development.

Additional signals between HERS cells and mesenchyme control root

elongation. Sonic hedgehog (SHH) expression within HERS cells is thought

to signal target genes Patched1 (Ptc1) and Gli1 and promote proliferation of

dental mesenchyme (Nakatomi et al., 2006). PTC mutants show reduced cell

division within dental mesenchyme, shortened tooth roots, and disturbances

in tooth eruption. Autocrine or paracrine expression of insulin‐like growth

factor‐1 (IGF‐1) by HERS cells has also been implicated in regulating root

elongation. IGF‐1 receptors are present on HERS cells, and application of

IGF‐1 to mouse HERS organ cultures, in vitro, resulted in elongation of the

root sheath, possibly from increased cell proliferation from the OEE

(Fujiwara et al., 2005).

Some enamel proteins, including amelogenins and ameloblastin, in addi-

tion to their roles in crown formation (Section II and Table I), have been

proposed as regulators of R/PT formation (Table II). Reports have indicated

expression of these molecules in the HERS region and in the pulp region,

although at low levels and with a great deal of variability between species

(Bosshardt and Nanci, 1997, 2004; Fong and Hammarstrom, 2000; Janones

et al., 2005; Nebgen et al., 1999; Oida et al., 2002; Papagerakis et al., 2003;

Zeichner‐David et al., 1997). Amelogenin KO mice exhibit a tooth pheno-

type resembling amelogenesis imperfecta (AI) in humans, namely hypoplas-

tic enamel characterized by poorly organized hydroxyapatite crystals,

resulting in chalky‐white, fragile teeth (Gibson et al., 2001a). Additional

studies of amelogenin KO mice have implicated a role for amelogenin in

root development or maintenance. Increased osteoclastic root resorption

(Hatakeyama et al., 2003) and decreased BSP expression (Viswanathan

et al., 2003) by root surface cells have been reported in amelogenin KO mice

compared to controls. It has been further proposed that amelogenin and the

alternatively spliced product LRAP may be involved in regulating levels of

receptor activator of NF‐�B ligand (RANKL) within the local tooth root

environment, thereby acting as a protector of against osteoclast‐mediated

resorption of the root surface (Hatakeyama et al., 2006). In amelogenin KO

mice, RANKL on the tooth root surface was increased, as determined by

immunohistochemistry. Binding of RANKL (secreted from osteoblasts,

PDL cells, cementoblasts, etc.) to RANK on osteoclast precursor cell sur-

faces results in maturation/activation to functioning osteoclasts. Further,

80 Foster et al.

it was reported that adding LRAP to cocultures of PDL/cementum cells and

bone marrow cells resulted in decreased RANKL expression. Boabaid et al.

additionally report that LRAP increased OPG and had a slight eVect (notstatistically significant) on decreasing RANKL expression in immortalized

cementoblasts, in vitro (Boabaid et al., 2004b).

The epithelial cell rests of Mallasez (ERM) that correspond with the

remnants of the root sheath within the periodontal ligament have been

known to express transcription factors and signaling molecules including

OPN, and BMP‐2 and 4 (Mouri et al., 2003; Rincon et al., 2005). These

findings have led some researchers to suggest that ERMs aVect periodontaltissues within the local environment (Table II). Additional evidence that

ERMs may have a role in repair of root tissues has been provided by

Hasegawa et al., who reported that in early stages of cementum repair and

adjacent to sites of root resorption, ERM cells express OPN, ameloblastin,

and BMP‐2, molecules associated with regulation of mesenchymal cell

behavior (Hasegawa et al., 2003).

While the potential role of epithelial products in root development

warrants further study, EmdogainÒ (Straumann Biologics, Waltham, MA,

USA), an epithelial protein derivative aimed at regenerating periodontal

tissues has been in use clinically for many years. EmdogainÒ, an extract of

porcine tooth germs, promotes periodontal regeneration with varied reports

of successful outcomes (Bartlett et al., 2006; Esposito et al., 2005; Giannobile

and Somerman, 2003; Heden and Wennstrom, 2006; Venezia et al., 2004).

In vitro studies have suggested that EmdogainÒ may preferentially promote

proliferation, matrix production, and diVerentiation/mineralization in PDL

fibroblasts (Gestrelius et al., 1997; Lyngstadaas et al., 2001), with increased

proliferation but varying influence on gene expression in dental follicle cells

(Hakki et al., 2001), osteoblasts, and putative cementoblasts (Tokiyasu

et al., 2000).

While the predominant protein in EmdogainÒ is amelogenin, several other

factors have been reported, including alternatively spliced and proteolytically

cleaved amelogenins, LRAP and TRAP respectively, as well as amelo-

blastin, TGF‐�, and BMPs (Kawase et al., 2001, 2002; Maycock et al.,

2002; Suzuki et al., 2005; Takayama et al., 2005). Both in vitro and in vivo

EmdogainÒ and the amelogenins have been confirmed to have bioactive

signaling properties (Bartlett et al., 2006; Boabaid et al., 2004b; Esposito

et al., 2005;Giannobile and Somerman, 2003; Swanson et al., 2006; Tompkins

and Veis, 2002; Tompkins et al., 2005; Veis, 2003; Venezia et al., 2004;

Viswanathan et al., 2003). Evidence for a signaling role for amelogenins has

been bolstered by identification on myoblast cells of an LRAP‐interactive cellsurface binding protein, lysosomal adhesion membrane protein‐1 (LAMP‐1)(Tompkins et al., 2006). Using a yeast two hybrid system, enamel matrix

proteins were also found to interact with a large number of secreted and


membrane proteins, notably LAMP‐3 (Wang et al., 2005a). Further studies of

these enamel matrix proteins will help to clarify the significance of these

putative receptors and interacting proteins, and subsequently elucidate their

signaling role, if any, in root/periodontal tissue formation.

B. Bone Morphogenetic Proteins

The importance of BMPs and BMP antagonists for contolling crown devel-

opment is well established (Bei et al., 2000; Ferguson et al., 1998; Iwata et al.,

2002; Kratochwil et al., 1996; Laurikkala et al., 2003; Maas and Bei, 1997;

Zhang et al., 2000). More recent studies have provided insights into the

interactions of various BMPs and their associated antagonists during bone

and crown development, for example, negative regulators ectodin and follis-

tatin. However, the significance of these interactions during root develop-

ment warrants further investigation (Kassai et al., 2005), especially with the

awareness that BMPs have very complex interactions in other tissues. For

example, BMP‐2, 4, 7 can induce ectopic bone formation individually, but

BMPs also form heterodimers, for example, BMP‐2/7, and 4/7, with greater

mineral stimulating ability than their constituents (Franceschi, 2005)

(Tables II and III).

As one approach to defining the role of BMPs in tooth development,

Plikus and colleagues evaluated the eVects of downregulating expression of

BMP signaling in oral and dental tissues by creating keratin 14‐Noggin

transgenic mice (Plikus et al., 2005). These mice developed a wide spectrum

of tooth phenotypes that included abnormal histogenesis and diVerentiationof ameloblasts and odontoblasts, a decrease in the number of teeth devel-

oped, reduction and/or alteration in size and shape of teeth, as well as

changes in the size and shape of roots. Root alterations included failure of

molar teeth to form multiple roots and lack of definition in the cementum‐enamel junction (CEJ) region. Using a similar strategy to better define the

role of gremlin, a BMP antagonist, in osteoblastic diVerentiation and func-

tion, Gazzero and colleagues generated mice with conditional gremlin over-

expression by employing an osteocalcin promoter driven—gremlin construct

(Gazzerro et al., 2005). Mice overexpressing gremlin exhibited an osteopenic

bone phenotype that included impaired bone formation, bone fractures,

disorganized collagen bundles at the endosteal cortical surface, a marked

decrease in osteoblast numbers, and reduced mineral apposition and bone

formation rates versus WT littermate controls. In addition, although not

detailed, incisor teeth were observed to be fragile in gremlin overexpressing

mice compared to WT controls.

BMP‐3 has emerged as a signaling factor that unlike other BMPs that

promote osteoblast/cementoblast, odontoblast diVerentiation, antagonizes

Table III Factors Associated with Reported Root Phenotypes


Periostin Preferentially expressed in

cells associated with bone,

lung, kidney, heart valve,

although found in many

other tissues, including

cancerous tissues. Also

expressed at high levels in

embryonic periosteum

Teeth: PDL region (restricted

to PDL after postnatal day

7 in mice)

Epithelia

Papillae cells

Odontoblasts

Follicle cells

Alveolar bone region

Periostin is a secreted 90‐kDa protein

with strong homology to the insect

cone guidance protein fasciclin I

family (includes �ig‐h3). Periostinis thought to be involved in cell

adhesion via �v�3 and �5 integrins,

although periostin does not contain

an RGD motif. Exact function for

this protein remains to be

established

Suggested general functions include:

(1) Induction of angiogenesis, (2)

Regulation of hard‐soft tissueinterfaces, (3) Regulation of

deposition and organization of

other ECM molecules, (4)

Protective function during stress/

mechanical load (e.g., in teeth:

chewing/tooth movement/ tooth

eruption), and (5) promotion of cell

migration and adhesion. Periostin

gene and protein are reported to be

induced by several factors including

BMP‐2, TGF‐�, PDGF, and

angiotensin II

In humans, five alternatively spliced

forms have been identified, but

functions not established

Periostin KO: exhibits a strong tooth/

periodontal phenotype: widening of

PDL; root resorption; increased

osteoclasts all suggestive of

aggressive periodontal disease.

Further, the incisors exhibit

compression of enamel and dentin

suggested to be related to a

proposed role for periostin in

controlling shear force/collagen

degradation

At birth, KO mice appear normal but

many die before weaning and those

that survive have growth

retardation. Trabecular bone is

decreased, although this phenotype

is not as dramatic as that of the

tooth. Partial correction of defects,

most notably incisor enamel, is

noted when the animals are given a

soft diet

Mice with periostin overexpression

exhibit cardiac dilation and

dysfunction

BMP‐4 KO mice have decreased levels

of periostin in mesenchymal tissues

and MSX2 KO mice have increased

evels of periostin

Gillan et al. (2002); Horiuchi

et al. (1999); Kii et al.

(2006) ; Kruzynska ‐ Frejtaget al. (2004); Li et al.

(2005a); Rios et al. (2005);

Suzuki et al. (2004); Wilde

et al. (2003)

NF1‐C/CTF (nuclear

factor I‐C)NF1 protein family

of site‐specificDNA‐bindingproteins (also

known at CTF or

CAAT box

transcription

factor)

General: found in many

tissues during development

and in mature tissues as

well

Tooth associated include:

dental papilla region

Ameloblasts Odontoblasts/

preodontoblasts (strong

expression during root

formation)

Mesenchymal cells

Stellate reticulum region

PDL region

Bone region

HERS

Suggested function: in NF1‐C KO

mice, HERS cells fail to proliferate

and/or fail to induce odontoblast

diVerentiation required for root

formation. However, the specific

transcription factors and cell‐signaling pathways disrupted in

cells from NF1‐C KO mice remain

to be defined

NF1 protein family: functions both in

viral DNA replication and in the

regulation of gene expression

NF‐1C KO: defective root

development

Steele‐Perkins et al. (2003,2005)

Incisors:

Maxillary: KO mice fail to form roots

but enamel and dentin appear

normal

Mandible: more severely aVected;

histologically, disorganized tissues

occur in place of incisors

Molars: crowns form, but no root

development is seen. Jaw bones

seem normal although during

preparation of heads for histology,

the teeth fall out and sockets are

shallow with an organized mesh of

bony spicules vs. WT tissues with

deep sockets and intact teeth. Also,

mandibles appeared approx. 10%

smaller vs. WT, but maxillary size

diVerences were not reported

Gene expression:

Noted decreased expression (50%) of

tooth‐associated genes dentin

sialoprotein, ameloblastin,

amelogenin in mandibles of KO vs.

WT; but normal transcripts for �1

type I col and Nfia, b, x

(Continued )

DMP‐1 (dentin

matrix protein‐1)Odontoblasts, osteoblasts,

osteocytes, hypertrophic

chondrocytes,

cementoblasts,

cementocytes, cementum

matrix, brain neurons,

other tissues (salivary

glands, muscle)

Role in mineral formation: dentin

matrix assembly and crystal growth

Multifunctional: attachment,

diVerentiation, activation of MMP‐9, role in osteocyte response to

mechanical stress

DMP‐1 KO:

Bone and cartilage: decrease in

mineral to matrix ratio, increase in

crystal size in bones, osteomalacia,

disordered canalicular network,

cartilage defect, chondrodysplasia‐like phenotype

Newborn DMP‐1 KO mice have

slightly expanded hypertrophic

zones and modest increase in bone

diameter vs. WT—DMP‐1 not

essential for early bone and tooth

development

Tooth: reduced dentin, increase in

predentin; reduced rate of dentin

formation; abnormal dentin

tubules; delay in/absent 3rd molars;

cementum—cementocytes not

healthy, but details on root defects

have not been reported

Overexpression of DMP‐1 in

pluripotent and mesenchymal cells

promotes an odontoblast

phenotype

Almushayt et al. (2006); Baba

et al. (2004b); Feng et al.

(2003); Foster et al. (2006) ;

Kalajzic et al. (2004); Ling

et al. (2005); Ye et al. (2005)

Table III Continued


BMP‐3 (bone

morphogenetic

protein 3)

Osteoblasts

Follicle cells

Cementoblasts

PDL cells

BMP‐2/4 are expressed in all tissues

involved in tooth formation, while

BMP‐3 expression is limited to the

follicle region and later in

development to the PDL region.

Existing data suggest that BMP‐3acts as an antagonist to BMP‐2/4.Based on this data it has been

suggested that BMP‐3 acts as a

regulator of soft–hard tissue

interfaces

BMP‐3 KO: increased bone mass,

however no specific tooth

phenotype has been reported

BMP‐3 overexpression: a 2006

abstract from Gagari et al. reported

decreased mass of dentin and

cementum, enlarged pulp

chambers, and widened PDL in

BMP‐3 transgenic mice vs. WT

littermate controls

Aberg et al. (1997); Chen et al.

(2004); Daluiski et al.

(2001); Gagari et al. (2006);

Gamer et al. (2005); Zhao

(2003)

BMPs/BMP

antagonists

All cells associated with tooth

development and

maturation

Known to have major roles in

controlling patterning and

diVerentiation of odontoblasts and

ameloblasts, BMPs have been

shown to enhance regeneration of

periodontal tissues (Nifusojeckii,

other reviews), but their specific

role during development of root/

periodontal tissues has not been

reported

Keratin14‐Noggin transgenic mice:

significant tooth phenotype with

alterations in number, size, shape,

and cell diVerentiation

Root specific: no mandibular molars,

maxillary molars—fail to form

multiple roots, poor or no CEJ,

small teeth with limited roots,

HERS forms, but proliferation of

cells in this region is limited

Osteocalcin‐gremlin transgenic mice:

General: impaired bone formation

and osteopenia

Tooth Phenotype: not explored in

depth but reported tooth fragility

Aberg et al. (1997); Gazzerro

et al. (2005) ; Nadiri et al.

(2004); Nifuji and Noda

(1999); Plikus et al. (2005);

Ripamonti (2005); Yanagita

(2005)

86 Foster et al.

these BMPs and so inhibits cementoblast and osteoblast function

(Bahamonde and Lyons, 2001). Gagari et al. (to date in abstract form only)

have reported that collagen type 1 promoter‐driven BMP‐3 transgenic mice

exhibited a tooth phenotype that included decreased cementum and dentin

mass, enlarged pulp chambers, and a widened PDL region (Gagari et al.,

2006). In contrast, BMP‐3 null mice have been reported to have decreased

bone density, but a tooth phenotype was not reported (Daluiski et al., 2001).

Additionally, Gamer and colleagues found that BMP‐3 interferes with both

activin and BMP signaling by binding to AcTRIIB, the common type II

receptor for BMPs (Gamer et al., 2005). These data, coupled with previous

studies demonstrating high expression for BMP‐3 in the follicle/PDL region,

support a role for BMP‐3 in regulating hard–soft tissue interfaces in the

periodontium (Aberg et al., 1997; Ripamonti and Reddi, 1997; Takahashi

and Ikeda, 1996; Yamashiro et al., 2003). The existing evidence is strong that

BMPs and associated antagonists are critical for R/PT development and

future studies in this area may contribute to novel therapies for regenerating

tooth structures.

C. Periostin and Nuclear Factor I‐C/CAAT Box Transcription Factor

While many of the molecules described in this section play a role in miner-

alized tissue development, two molecules that may have a more specific

critical role in root/periodontal tissue development have been identified from

their respective KOmouse phenotypes, which were remarkable for the condi-

tion of their periodontia. These molecules include the extracellular matrix

protein, periostin, and the nuclear factor I‐C/CAAT box transcription factor,

NFI‐C/CTF.Periostin has been detected in many tissues (Nakamura et al., 2005), but

mice null for periostin exhibit a very specific tooth phenotype (Rios et al.,

2005) (Table III). Mice lacking the periostin gene appear relatively normal at

birth but develop a condition resembling an aggressive form of periodontal

disease by 3 months of age. The defect seems to be selective to the root/

periodontal region, with odontoblasts and dentin only mildly altered when

compared with the periodontal apparatus. Some enamel defects are obser-

ved, but are limited to incisors and suggested to be associated with increased

enamel stress due to a weakened PDL (Rios et al., 2005), or possibly due to

disruption of the shear zone important for continuously erupting teeth (Kii

et al., 2006). These data suggest the periostin may play a key role in protecting

the root surface from root resorption, as well as for maintaining a functional

periodontal ligament. The knowledge gained from continued studies directed at

defining the factors regulating periostin expression during root formation, and

the relationship between periostin and other root/PDL‐associated extracellular


matrix molecules should prove valuable long‐term for designing regenerative

therapies.

The nuclear factor I‐C/CAAT box transcription factor (NFI‐C/CTF) KO

mice have provided new insights into molecules that may be involved in

directly regulating root development. There are four genes encoding nuclear

factor I transcription‐replication proteins in mammals, NFI‐A, ‐B, ‐C, and ‐X.

Members of the NFI protein family of site‐specific DNA‐binding proteins

function both in viral DNA replication and in the regulation of gene expres-

sion. NFI‐C/CTF contains a prototypical proline‐rich transcription activation

domain and a heptamer repeat that is homologous to the C‐terminal domain

of RNA polymerase II (Gronostajski, 2000).

To elucidate the physiological roles for this family of nuclear transcription

factors, Gronostajaski’s laboratory has focused on disrupting their expres-

sion (Steele‐Perkins et al., 2003, 2005). Although NFI‐C is expressed in

many organ systems, including developing teeth, disruption of the Nfic gene

in mice resulted primarily in a unique tooth phenotype: molars lacking roots,

thin and brittle mandibular incisors, and weakened and abnormal maxillary

incisors. Molar crown development is normal and animals on a soft diet are

fertile and live as long as their littermates (Steele‐Perkins et al., 2003).

D. Regulators of Phosphate and Pyrophosphate Metabolism

1. Progressive Ankylosis Protein, Plasma Cell Membrane Glycoprotein 1,

and Tissue Nonspecific Alkaline Phosphatase

Regulators of phosphate metabolism have received considerable attention

within the last 5 years, with convincing evidence that inorganic phosphate

(Pi), beyond its known role as an important component of hydroxyapatite

mineralization, may also regulate cell behaviors and mineralization as a

signaling molecule. Conversely, pyrophosphate (PPi) functions as a well‐known and potent inhibitor of hydroxyapaptite formation. Table IV pro-

vides information on the role of Pi and PPi associated genes and their protein

products in regulating mineralized tissues. These include mouse progressive

ankylosis gene (ANK, as well as human homolog, ANKH), a putative

transporter of PPi from the intracellular compartment to the extracellular

space (Ho et al., 2000), the PPi‐generating nucleoside triphosphate pyropho-

sphohydrolase plasma cell membrane glycoprotein‐1 (PC‐1) (Goding et al.,

1998), and tissue nonspecific alkaline phosphatase (TNAP), an enzyme

proposed to cleave PPi substrate to its Pi constituents (Whyte et al., 1995).

Further information regarding the functions of these proteins is provided in

Table IV, including implications of their roles based on mouse models, for

example, mutation or KO. Results from studies to date suggest that local

Table IV Regulators of Phosphate (Pi) and Pyrophosphate (PPi) Metabolism

Factor Cells Function/Putative Function Models References

PC ‐1: (NPP1—nucleotide

pyrophosphatase

phosphodiesterase ‐1)gene symbol: Enpp1

ANK: proteins of mouse

progressive ankylosis

(ank) gene. (ANKH,

human homologue) gene

symbol: Ank

Expressed by a diverse

group of cells including

osteoblasts, PDL cells,

odontoblasts, follicle

cells, and

cementoblasts, among

others

PC‐ 1: increases intra/extracellular and matrix

vesicle PPi ; inhibits apatite

deposition

ANK: transporter/

cotransporter of PPi from

intracellular to

extracellular matrix;

inhibits apatite deposition

PC ‐1/ANK mutations:

In animals/humans with mutations in

these genes, ectopic calcification and

decreased extracellular PPi occur. In

murine models with mutations in either

PC‐ 1 or Ank, there is a marked increase

(� 10‐ fold) in cementum formation

(appears to be cellular), while dentin,

enamel, PDL region, and surrounding

alveolar bone appear normal (Fong et al.,

2005; Nociti et al., 2002). There have been

no reports of a tooth phenotype in humans

Fedde et al. (1999); Fong

et al. (2005) ; Harmey et al.

(2004); Ho et al. (2000);

Johnson et al. (2003);

Nociti et al. (2002);

Nurnberg et al. (2001) ;

Okawa et al. (1998);

Reichenberger et al. (2001) ;

Rutsch et al. (2000); Rutsch

et al. (2001); Terkeltaub

(2001)

TNAP: (TNSALP, ALKP,

ALK) tissue nonspecific

alkaline phosphatase

gene symbol: Akp2

Expressed by a diverse

group of cells including

osteoblasts, PDL cells,

odontoblasts, follicle

cells, and

cementoblasts, among

others

A marker of cell (osteoblast/

cementoblast)

di V erentiation; catalytic

function in mineralization;

may transport ions across

membrane; hydrolyzes the

mineralization inhibitor,

PPi

TNAP mutations: Chapple (1993); Beertsen

et al. (1999); van den Bos

et al. (2005); Whyte, (2002)

In animals/humans there are a variety of

forms of TNAP‐associatedhypophosphatasia, which result in locally

increased levels of PPi and osteopenia. In

the area of the tooth root, cementum

deficiency severely compromises anchoring

of the PDL between bone and the tooth,

with severe periodontal disease and

eventually tooth loss

TNAP KO: appears to aVect cementum

formation selectively vs. dentin/enamel.

There is no or minimal cementum

formation and hence no PDL attachment

and teeth are exfoliated


control of PPi/Pi is critical for normal root/periodontal tissue development,

and further that cementum may be a uniquely sensitive tissue to PPi and Pi in

the local area. In cases of TNAP deficiency (TNAP mutation or KO, the

condition hypophosphatasia in humans), bones are osteopenic and root

cementum is disrupted, generally with a lack of acellular cementum and

severely disrupted cellular cementum (Beertsen et al., 1999; van den Bos

et al., 2005). Lack of cementum prevents insertion of PDL (Sharpey’s) fibers,

leading to lack of attachment and exfoliation of teeth. In contrast, humans

and animals with loss of function of PC‐1 or ANK exhibit low levels of PPi

in the local extracellular environment, resulting in ectopic calcifications in

joints, with mice exhibiting an arthritis‐like condition (Ho et al., 2000;

Terkeltaub, 2001). Humans withmutations in these genes also present pathol-

ogies resulting from deficient PPi, including craniometaphyseal dysplasia

(CMD) and idiopathic infantile arterial calcification (IIAC) (Nurnberg

et al., 2001; Reichenberger et al., 2001; Rutsch and Terkeltaub, 2005; Rutsch

et al., 2001). An unexpected and intriguing tooth phenotype has been reported

inmice withmutations in either PC‐1 or ANK. Rather than observing ectopic

calcification in the PDL, a marked increase in cementum formation was

observed, while PDL, dentin, and alveolar bone appeared unaVected (Nociti

et al., 2002). Ongoing studies are directed at examining the mechanical and

structural properties of all of these mineralized tissues, under situations in

mice, where PPi and/or Pi have been altered.

The importance of maintaining the appropriate concentration of Pi in the

extracellular environment for regulation of mineralization was highlighted

by the elegant studies of Murshed and colleagues (Murshed et al., 2005).

By studying a variety of KO mice, the critical nature of modulating extra-

cellular Pi concentration both for regulating physiological mineralization

and preventing pathological calcification was described. However, details

on R/PT regulation and development were not included, and require further

analysis using the animal models developed by this group. Studies focusing

on teeth and surrounding regions may provide insight into mechanisms

leading to altered cementum versus apparently normal enamel and dentin

phenotypes in cases of altered Pi/PPi homeostasis.

In addition to ANK, PC‐1, and TNAP, several other Pi‐regulating pro-

teins have been demonstrated to be important in controlling mineralization,

although tooth phenotypes in some cases have not yet been reported. These

are described in the following sections.

2. Phosphate‐Regulating Gene with Homology to Endopeptidase on the

X‐Chromosome

X‐linked hypophosphatemic rickets (XLH/HYP), the most common form of

rickets in humans, is caused by a mutation in the PHEX gene. The murine

90 Foster et al.

homologue (Hyp mice) of the human disease is marked by renal phosphate

wasting, abnormal regulation of vitamin D metabolites, rickets, osteomala-

cia, growth retardation, resistance to vitamin D therapy, hypophosphate-

mia, and high levels of FGF‐23 and MEPE (Quarles, 2003; Rowe, 2004).

Additionally, cartilage abnormalities were reported in Hyp mice, resulting

potentially from participation of PHEX in regulation of growth plate carti-

lage (Miao et al., 2004). Dentin and bone hypomineralization in Hyp mice

may result from not only low serum Pi, but also some intrinsic osteoblast/

odontoblast defect (Ogawa et al., 2006). A tooth root phenotype has not

been reported.

3. Fibroblast Growth Factor 23

FGF‐23 regulates phosphate homeostasis, and FGF‐23 mutation in humans

causes autosomal dominant hypophosphatemic rickets (ADHR) (Rowe,

2004; White et al., 2006; Yu and White, 2005). FGF‐23 null mice exhibit

growth retardation, hyperphosphatemia, increased levels of 1, 25 vitamin D

levels, increased total‐body Bone Mineral content but decreased Bone

mineral density of limbs, and premature death by 13 weeks of age (Razzaque

et al., 2006; Sitara et al., 2004). Although a general increase in mineralization

resulted, there was also an accumulation of unmineralized osteoid associated

with limb deformities and excessivemineralization of soft tissues such as heart

and kidney. Crossing of FGF‐23 null with Hyp mice (PHEX mutation,

equivalent to X‐linked hypophosphatemia) resulted in a mouse phenotype

resembling the FGF‐23 null in skeletal phenotype and serum phosphate,

suggesting that FGF‐23 is upstream of PHEX (Sitara et al., 2004). There

has been some evidence for PHEX involvement in degradation of FGF‐23(Bowe et al., 2001), and results from the Hyp/FGF‐23 null mice are consistent

with the hypothesis that increased FGF‐23 in PHEX mutated mice and

humans may be responsible for the observed Pi disorder (Sitara et al., 2004).

4. Matrix Extracellular Phosphoglycoprotein

Matrix extracellular phosphoglycoprotein, a member of the SIBLING ex-

tracellular matrix protein family (Fisher and Fedarko, 2003) is expressed by

several mineralized tissue‐associated cells, including osteoblasts and osteo-

cytes, hypertrophic chondrocytes, dental pulp cells, and odontoblasts, as

well as other tissues (Argiro et al., 2001; Liu et al., 2005b; Lu et al., 2004;

MacDougall et al., 2002; Nampei et al., 2004; Rowe et al., 2000). Increased

expression of MEPE protein has been noted in humans with XLH and their

Hyp mouse counterparts (Argiro et al., 2001; Guo et al., 2002; Liu et al.,

2005c; Rowe, 2004). MEPE is thought to control renal phosphate excretion

and to modulate mineralization. MEPE contains an acidic serine aspartic


rich motif, ASARM, that is cleaved by cathepsin B, and PHEX inhibits this

activity (Rowe et al., 2004). One hypothesis is that the ASARM motif

inhibits crystal growth, with MEPE KO mice therefore exhibiting acceler-

ated mineralization and bone formation. Interestingly, it was reported that

dentonin, a fragment of MEPE isolated from erupted human molars pro-

moted the proliferation of dental pulp stem cells, in vitro, and it was specu-

lated that such molecules may have potential to participate in repair of lost

or damaged dentin (Liu et al., 2004).

E. Factors Known to Regulate Osteoprogenitor Cells and Osteoblasts

1. Wnt, Hedgehog, Osterix, and Nuclear Factor of Activated T Cells

Pluripotent mesenchymal stem cells (MSCs) have the potential to diVerenti-ate into several diVerent cell types, and specific transcription factors that

have been found to commit MSC diVerentiation to the osteoblast lineage

require further study for their potential role in root and periodontal tissue

development. Sequential expression of Indian hedgehog (Ihh) and canonical

Wnt signals at progressive stages of osteoblast development has been found

to coordinate the expression of transcription factors directing osteoblast

diVerentiation (Hu et al., 2005). For example, osterix (Osx) regulates down-

stream genes that commit MSCs to an osteoblast lineage, while NFAT was

found to cooperate with Osx to accelerate osteoblast diVerentiation and

bone formation (Koga et al., 2005; Tai et al., 2004, 2005). Further, the expres-

sion of p53 had been found to repress Osx expression, inhibiting osteoblast

diVerentiation and favoring osteoblast contributions to osteoclastogenesis

(Wang et al., 2006) (Table V).

2. Runt‐Related Transcription Factor 2 and TaVazin

Runt‐related transcription factor 2 (Runx2) expression is also necessary for

osteoblast diVerentiation and function and a role in developing tooth crown

has been identified (Aberg et al., 2004a,b; D’Souza et al., 1999). In osteo-

blasts, Runx2 directly stimulates transcription of osteoblast‐related genes

such as OCN, type I collagen, OPN, and collagenase type III (Ducy et al.,

1997; Franceschi et al., 2003; Kern et al., 2001). Canonical Wnt signaling

upregulates Runx2 expression (Gaur et al., 2005), and Runx2 subsequently

coordinates diverse signals involved in osteoblast diVerentiation and activity

(Franceschi et al., 2003). For example, the transcription factor, taVazin(TAZ), is an endogenous co‐activator of Runx2 in cells, and therefore an

endogenous regulator of osteoblast diVerentiation (Cui et al., 2003). Inter-

estingly, TAZ simultaneously represses gene transcription associated with

92 Foster et al.

the adipocyte diVerentiation pathway (Hong et al., 2005). Stimuli that

promote bone formation via regulation of transcription have been found

to upregulate both TAZ and Runx2 (Hong et al., 2005). Runx2 expression

during tooth development also has several tooth‐specific downstream targets

(Gaikwad et al., 2001). The formation of successional teeth is inhibited by

Runx2 activity (Wang et al., 2005c), and during tooth morphogenesis Runx2

mediates FGF signaling between epithelium and mesenchyme (Aberg et al.,

2004b). Runx2 has also been identified in periodontal ligament cells; however,

its function appears to be suppressed, preventing diVerentiation of PDL cells

toward osteoblasts (Saito et al., 2002).

3. Activating Transcription Factor 4

Activating transcription factor 4 (ATF4) was identified as a factor for

osteoblast diVerentiation, and it is in turn the substrate for p90 ribosomal

S6 kinase 2 (RSK2), a growth factor‐regulated kinase (Yang et al., 2004).

A mutation in RSK2 was mapped as the cause for CoYn‐Lowry Syndrome

(CLS), which is associated with skeletal abnormalities in addition to mental

retardation. Mice null for ATF4 displayed a phenotype indicative of osteo-

blast defects, namely delayed bone formation in early development and low

bone mass. In addition to regulating type I collagen, ATF4 was found to act

cooperatively with Runx2 in regulating the OCN promoter in osteoblasts

(Xiao et al., 2005). No details on a tooth phenotype were provided.

4. Receptor Activator of NF‐kB Ligand and Osteoprotegerin

Receptor activator of NF‐�B ligand (RANKL) and osteoprotegerin (OPG)

have emerged as the primary factors in the axis of regulation of osteoclasts

and their precursors (Takahashi et al., 1999; Tsuda et al., 1997), and parallel

roles in tooth development and eruption have been described (Ohazama

et al., 2004a; Rani and MacDougall, 2000; Wise et al., 2002). Little is known

of the role of cementoblasts in osteoclast‐mediated turnover. Cementoblasts,

as well as cells of the nearby PDL and their precursors, express RANKL and

OPG (Boabaid et al., 2004a; Liu et al., 2005a; Sakata et al., 1999) and so may

be considered to take part in the regulation of osteoclastogenesis, though it is

currently not clear how these tissues participate in conditions of health and

disease. Cementum itself does not undergo significant physiological remo-

deling unlike the nearby alveolar bone that undergoes rapid turnover

throughout life. Additionally, though cementum resorption is not unknown,

it is relatively infrequent compared to osteoclast‐mediated resorption of

bone. Even in advanced periodontal disease, alveolar bone may be severely

eroded while cementum remains intact. This indirect evidence supports a pro-

tective role for cementum against biological resorption, a hypothesis supported


by results from in vitro (Boabaid et al., 2004a; Hatakeyama et al., 2006; Nociti

et al., 2004) and in vivo investigations (Hatakeyama et al., 2003). Further study

of the involvement of cementoblasts and PDL in osteoclastogenesis should

elucidate their role in osteoclastogenesis in the periodontium.

5. Dlx Transcription Factors

Members of the Dlx family of transcription factors, a subfamily of divergent

homeobox genes related to the Drosophila distal less (Dll) gene, have been

implicated as key regulators of tissue development and cell diVerentiation(Stock et al., 1996). Currently, six Dlx genes have been identified, in both

humans and mice, with convincing evidence that they play critical roles

in diVerentiation of bone‐forming cells. Dlx KO mice have profound cranio-

facial defects and absence of molars (Thomas et al., 1997). In humans,

tricho‐dento‐osseous (TDO) syndrome, characterized by enamel defects,

enlarged pulp chambers, and distorted roots (taurodontism), has been linked

to a mutation in Dlx3 (Price et al., 1998). Interestingly, in a very preliminary

study, Morsczeck reported that Dlx3 increased in dental follicle cells during

‘‘osteogenic’’ diVerentiation, in vitro.

F. Emerging and Other Factors to Consider

1. Proteoglycans

Another important group of molecules present in cementum and known to

play critical roles in tooth development are the proteoglycans (PGs). Proteo-

glycans are macromolecules composed of core proteins and glycosaminogly-

cans (GAGs). Small leucine rich proteoglycans (SLRPs) including decorin,

biglycan, lumican, osteoadherin/osteomodulin, and fibromodulin have been

suggested to play important roles in collagen‐linkedmineralization (Buchaille

et al., 2000; Couble et al., 2004; Embery et al., 2001; Iozzo, 1998). The im-

portance of PGs for appropriate crown development has been highlighted

(Goldberg et al., 2005) and indicates that further studies are warranted to

determine their respective roles during root formation. Several studies have

implicated SLRPs as being important for controlling mineralization of dental

tissues, and it was demonstrated that defective enamel and dentin formation

resulted from loss of biglycan and decorin in KOmice (Goldberg et al., 2005).

Although roles for PGs in root development have not been clarified, SLRPs

are apparently present in all mineralized tissues, and it can therefore be

safely asserted that there is some important, conserved function for this class

of PGs in mineralized tissues. Immunohistochemical studies have shown that

94 Foster et al.

cementum in several species (rat, human, mouse) is immunoreactive to various

PGspecies (Yamamoto et al., 2004b). PGpresence and involvement in develop-

ment was further supported in a rat molar model by intense immunoreactivity

for chondroitin 4‐sulfate, chondroitin 6‐sulfate, and unsulfated chondroitin,

during early phases of a cellular cementogenesis. GAGs and PGs have been

detected inmature PDL, and taken together these data provide strong evidence

that PGs are important for regulating collagen fibril formation during cemen-

togenesis and in the mature periodontium (Buchaille et al., 2000; Hakkinen

et al., 1993, 2000; Kaneko et al., 2001; Matias et al., 2003).

2. Small Integrin‐Binding Ligand N‐Linked Glycoprotein Family

The SIBLING (Small Integrin‐Binding Ligand N‐Linked Glycoprotein)

family is composed of genes located on human chromosome 4 (mouse

chromosome 5) encoding noncollagenous extracellular matrix‐associatedproteins associated with bones and teeth. While sequence homology among

SIBLINGs is not high, their relatedness is suggested by common organiza-

tional features and similar, functionally important post‐translational mod-

ifications (Fisher and Fedarko, 2003; Huq et al., 2005; Qin et al., 2004).

Genes in this family include bone sialoprotein (BSP), osteopontin (OPN),

dentin matrix protein‐1 (DMP‐1), dentin sialophosphoprotein (DSPP), and

matrix extracellular phosphoglycoprotein (MEPE). The DSPP transcript is

processed and results in two proteins, namely dentin sialoprotein (DSP) and

dentin phosphoprotein (DPP). SIBLINGs have common sequences includ-

ing an arginine‐glycine‐aspartate (RGD) integrin‐binding domain that likely

functions in signaling and cell attachment (Ganss et al., 1999; Sodek et al.,

2000), and an ASARM or similar motif (in all except BSP) that may be a

mineral inhibitory domain. SIBLINGs typically undergo extensive post‐translational modification, including enzymatic cleavage (in DSPP and

DMP‐1), phosphorylation, glycosylation, and likely more complex proces-

sing such as polymerization, in vivo (Gericke et al., 2005; He et al., 2005b;

Kaartinen et al., 2005; Qin et al., 2004). Several lines of evidence support a

role for SIBLINGs in root development (Bosshardt, 2005; Diekwisch, 2001;

Saygin et al., 2000), including the timed and spatial expression of BSP and

OPN during development and repair of root/periodontal tissue (Bosshardt

et al., 1998; D’Errico et al., 1997; MacNeil et al., 1994; Shigeyama et al.,

1996), coupled with their suggested roles in nucleation and regulation of

crystal growth (Boskey et al., 2000; Gericke et al., 2005; He et al., 2005a;

Tartaix et al., 2004). However, mice null for OPN, BSP, or MEPE have not

been reported to exhibit a root/periodontal phenotype, suggestive of some

redundancy with other molecules. DMP‐1 and DSPP, originally identified in

dentin and thought to be specific for this tissue, have been shown to not only

be critical for dentin development, but also present in other mineralized

Table V Factors Known to Regulate Osteoprogenitor cells and Osteoblasts (Role in Cementogenesis Unknown)


Wnt: wingless int

Hh: hedgehog

Ihh: Indian

hedgehog

Mesenchymal stem cells

(MSCs), many others

Osteoblast diVerentiation; Hh and

Wnt signals control osteoblast

development in a sequential

manner

Ihh is expressed in prehypertrophic

and early hypertrophic

chondrocytes and signals to

immature chondrocytes and

perichondrial cells. Canonical Wnt

signaling is downstream of Ihh

signaling

During tooth development Hh and

Wnt signals are emitted from the

cap stage enamel knot and are

considered to have a role in crown

morphogenesis

Many participants in the Wnt pathway

have been associated with mineralized

tissues, in health and disease:

Dickkopfs (Dkks), secreted frizzled‐related proteins (sFRPs), Wnt

inhibitory factor 1 (Wif1), LDL

receptor protein 5 (LRP5), and Wnts

4, 10b, and others

Ihh KO:

Shows dysregulation of chondrocyte

maturation and absence of expression

of target genes for the Wnt canonical

pathway in the perichondrium

Lrp5 KO:

Lrp5 is a transmembrane protein that

forms part of the cell surface receptor

complex binding Wnt within the Wnt

canonical pathway. Lrp5 deficient

mice exhibit osteopenia with fewer

total osteoblasts per bone area, and a

50% reduction in bone formation

Canalis et al. (2005); Hu et al.

(2005) ; Kato et al. (2002);

Li et al. (2005b); Nusse

(2005); St ‐Jacques et al.(1999); Thesle V et al.

(2001); Vaes et al. (2005);

Westendorf et al. (2004)

Osx: osterix

NFAT: nuclear

factor of activated

T cells

MSCs, osteoblasts Osterix is a transcription factor

required for osteoblast

diVerentiation, operating

downstream of Runx2; NFAT

cooperates with Osx to accelerate

osteoblast diVerentiation and bone

formation

Osx null mice:

Homozygotes for the mutation are not

viable, and lack both endochondral

and intramembranous bone

formation; developing tooth germs

appear unaVected

NFAT KO:

Mice are embryonically lethal

Koga et al. (2005) ;

Nakashima et al. (2002);

Tai et al. (2005)

(Continued )

x2: runt‐relatedanscription

ctor 2

In bone, MSCs; in

developing teeth: dental

mesenchyme, including

papilla, follicle cells,

and periodontal

ligament during pre‐eruptive tooth

development

Transcription factor necessary for

osteoblast diVerentiation; focal

point for integration of a variety of

signals aVecting osteoblast activity

Runx2 KO:

Functional osteoblasts, mineralized

bone, and hypertrophic cartilage are

absent

Tooth morphogenesis arrested in the

transition from the bud to cap stages

Aberg et al. (2004a,b) ;

Bronckers et al. (2001);

D’Souza et al. (1999);

Franceschi and Xiao

(2003); Franceschi et al.

(2003); Komori et al.

(1997); Otto et al. (1997)

Mediates epithelial‐mesenchymal

interactions during tooth

development

Z: TaVazin MSCs Acts as a transcriptional modulator

during osteoblast diVerentiation;

endogenous coactivator of Runx2;

promotes osteoblast formation,

inhibits adipocyte formation

No mouse models reported. In humans,

mutations in the TAZ gene are

responsible for Barth’s syndrome

(BTHS), X‐linked endocardial

fibroelastosis (EFE), X‐linked fatal

infantile dilated cardiomyopathy

(CMD3A), and familial isolated

noncompaction of left ventricular

myocardium (INVM)

Brady et al. (2006); Hong and

YaV e (2006); Hong et al.

(2005)

F4 activating

anscription

ctor 4

Osteoblasts ATF4 is a transcription factor that

regulates osteoblast diVerentiation

and function; cooperates with

Runx2 in stimulating osteoblast‐specific Ocn expression

ATF4 KO:

Delayed osteoblast diVerentiation

throughout the skeleton, and a

reduction in the area of mineralized

tissue visible in frontal and parietal

bones, clavicles and long bones; no

tooth phenotype reported

Xiao et al. (2005); Yang et al.

(2004)

le V Continued

tor Cells/Tissues Function/Putative Function Models References

Run

tr

fa

TA

AT

tr

fa

Tab

Fac

RANKL:

Receptor activator

of NF‐�B ligand

RANKL:

Osteoblasts,

cementoblasts, PDL

cells, dental follicle

cells, and many others

(e.g., cells of immune

system)

In bone RANK/RANKL receptor/

ligand expression in osteoblasts

promotes osteoclastogenesis; OPG

operates as a decoy receptor for

the RANK receptor, and inhibits

osteoclastogenesis

RANKL KO:

Severe osteopetrosis and lack of

osteoclasts, absence of tooth eruption

Amizuka et al. (2003); Bucay

et al. (1998); Katagiri and

Takahashi (2002); Kong

et al. (1999); Ohazama

et al. (2004a); Yasuda et al.

(1998); Yao et al. (2004)

Osteoprotegerin OPG:

Osteoblasts, PDL cells,

dental follicle cells,

dental epithelial cells,

dental papilla cells, and

many others (e.g., cells

of immune system)

Expression in dental tissues may

coordinate bone and tooth

development

OPG KO:

Increased osteoclastic activity and

bone remodeling, severe bone loss,

destruction of growth plate cartilage

and increased vascular calcification

OPG overexpression:

Osteopetrosis with normal tooth

eruption

p53 MSCs Osteoblast/osteoclast diVerentiation

(negatively regulates osteoblast

diVerentiation/function by

repressing expression of Osx), p53

deficiency confers osteoblasts with

an increased ability to promote

osteoclastogenesis

p53 KO:

Largely viable with a small proportion

with defects in neural tube closure.

Early onset of tumors, such as

lymphomas and sarcomas. High bone

mass, increased Osx expression, more

rapid diVerentiation in osteoblasts,

increased tendency for osteoblasts to

promote osteoclastogenesis

Armstrong et al. (1995);

Attardi and Donehower

(2005); Sah et al. (1995);

Wang et al. (2006)

98 Foster et al.

tissues and cells, including cementum (Baba et al., 2004a,b; Massa et al.,

2005; Qin et al., 2002, 2003). Cementum/root phenotypes have been indi-

cated in mice null for DMP‐1 and DSPP, but at present phenotypes have not

been fully reported (Sreenath et al., 2003; Ye et al., 2004). See Table III for

more details on the DMP‐1 deficient mouse model.

SIBLING family members also share a tendency for an astonishing degree

of multifunctionality, perhaps best embodied by the ubiquitous OPN.

In addition to well‐established roles as mineral regulators, SIBLINGs have

been identified as regulators of matrix metalloproteinase (MMP) function.

The MMPs are an extensive family of secreted or cell surface enzymes that

are critical in physiological development and remodeling of the extracellular

matrix, as well as certain pathologies. Several MMPs have been specifi-

cally associated with tooth development and remodeling (Apajalahti et al.,

2003; Bourd‐Boittin et al., 2005; Fanchon et al., 2004; Goldberg et al., 2003;

Maruya et al., 2003; Randall and Hall, 2002; Takahashi et al., 2003; Tsubota

et al., 2002), as well as periodontal and other oral diseases (Sorsa et al.,

2004). SIBLING proteins, BSP, OPN, and DMP‐1 were shown to specifically

activate MMP‐2, MMP‐3, and MMP‐9, respectively, even in the presence of

tissue inhibitors ofMMPs (TIMPs) (Fedarko et al., 2004). The significance of

SIBLING–MMP interaction in bone and tooth development is not yet clear,

but a model for localized interaction of matrix proteins and enzymes in tooth

development and eruption will surely be an important subject for further

study. An additional function for DMP‐1 as an intracellular transcriptional

regulator involved in diVerentiation has been proposed, indicating additional

mechanisms for SIBLING influence on tooth formation (Almushayt et al.,

2006; Narayanan et al., 2003).

3. Cementum Protein‐23

Cementum protein 23 was identified as a potential cementum marker by

screening a human cementum tumor cDNA library (Alvarez‐Perez et al.,

2006). However, cementum tumors are reported to exhibit a mixed cell type

so it is diYcult to determine the specificity of this protein/gene to cementum/

cementoblasts. Antibodies made to CP‐23 cross‐reacted with a cartilage type

collagen, type X collagen. CP23 has been identified in cementum, subpopu-

lations of cells in the PDL region, and specifically in PDL cells located

around blood vessels.

4. Betaig‐h3

Betaig‐h3 (�ig‐h3) is a collagen‐associated protein containing an RGD motif

that has been identified in several tissues and cells (Ohno et al., 2002). High

concentrations are found in cartilage and in the PDL region. A function


proposed is a negative regulator of osteogenesis, acting to maintain a struc-

tural balance between PDL and bone‐tooth interface. Further, there is

evidence for mechanical induction of �ig‐h3 and potential for this protein

to regulate chondrocyte diVerentiation via the TGF‐� pathway (Doi et al.,

2003; Ohno et al., 2005).

5. Brain‐Derived Neurotrophic Factor

BDNF, a member of the neurotrophin family, is considered to play a role in

survival and diVerentiation of central and peripheral neurons (Ebendal,

1992). In addition to being expressed in neural cells, BDNF is found in

many non‐neural cells/tissues including tooth germ, mature PDL, bone,

cartilage, heart, spleen, placenta, osteoblasts, immune cells, prostate, and

kidney (Nakanishi et al., 1994; Nosrat et al., 1998; Yamashiro et al., 2001).

Takeda et al., using a dog model of periodontal disease, reported that

BDNF promoted periodontal regeneration, that is, new bone, connective

tissue fibers, and new cementum (Takeda et al., 2005).

6. Bono 1

Bono 1 has been identified in bone cells, in secretory odontoblasts coexpressed

with DSPP, but not in pre‐secretory ameloblasts (where one does see DSPP)

and follicle cells (James et al., 2004). Bono 1 is associated with regions of

mineralization in bone, dentin and cementum, leading James et al. to propose

involvement in controlling mineral formation.

7. Connective Tissue Growth Factor

Connective tissue growth factor (36–38 kDa) (Asano et al., 2005; Shimo et al.,

2002; Yamaai et al., 2005) belongs to CCN family, that is, CTGF, CEF10,

and Nov. CTGF is found in several cells, including PDL cells, fibroblasts,

chondrocytes, dental mesenchyme cells, epithelial cells, vascular endothelial

cells of the enamel knot, pre‐ameloblasts and dental lamina (Friedrichsen

et al., 2003, 2005; Nakanishi et al., 2001). Studies to date indicated that

expression of CTGF is regulated by TGF‐�1/BMP‐2. Interestingly, CTGF

promotes expression of gene/proteins associated with PDL homeostasis, for

example, increased expression of type I collagen, periostin, andALP, but with

no eVect on OPN or OCN gene expression (Asano et al., 2005). Other studies

indicate that CTGF is involved in a chemotactic and mitogenic eVect in

fibroblast‐like cells in vitro, and further enhances cell proliferation andmatrix

synthesis in connective tissues linked towound healing (Lin et al., 2003, 2005).

KO animals have abnormal growth plates, while CTGF mutant mice have

impaired endochondral ossification (Ivkovic et al., 2003), though no tooth

100 Foster et al.

phenotype has been reported. Other studies using tooth germ implants in vitro

with CTGF antibodies report severe inhibition of proliferation of both

epithelial and mesenchymal cells, and a delay in cytodiVerentiation of amelo-

blasts and odontoblasts (Shimo et al., 2002). Intriguingly, CTGF is not

expressed in Cbfa1‐null mice embryos (Yamaai et al., 2005). Based on these

studies, there is growing evidence that CTGF may have a significant role in

the development of mineralized tissues and in promotion of endochondral

ossification.

8. Ectodysplasin (Tabby/Downless)

Ectodysplasin is associated with ectodermal tissues (Pispa and ThesleV, 2003;Sharpe, 2001). The tabby gene (Ta) encodes the soluble tumor necrosis factor

(TNF) ligand ectodysplasin (Eda). Eda binds to the TNF receptor (EdaR),

encoded by the downless gene (dl), and this interaction leads to NF�Bactivation via the cytoplasmic death domain adapter, Edaradd, encoded by

the crinkled locus gene (Cr) (Courtney et al., 2005). Mice with a mutation in

Ta, dl, or Cr display an ectodermal dysplasia phenotype characterized by

abnormal development of ectoderm derived structures, including teeth

(Courtney et al., 2005; Drogemuller et al., 2001; Risnes et al., 2005; Tucker

et al., 2000). Additionally, studies manipulating levels of signaling molecules

in the Eda axis support a critical role for these signals in determining tooth

shape and cusp number (Kangas et al., 2004; Pispa et al., 2004). Altered

signals from the enamel knot have been demonstrated in mutant mice, and

the most dramatic defects are seen in molars with reduced size and abnormal

shape (including roots). Specific root/PDL targeted signals related to these

genes (Ta, dl or Cr) have not been identified.Humanswithmutations in EDA,

EDAR or EDARADD genes have hypohidroitic ectodermal dysplasia, with

more markedly aVected individuals exhibiting severe tooth deformities and

tooth loss (Courtney et al., 2005).

9. Osteocrin

Osteocrin was initially identified using a virus‐based signal‐trap proteomic

approach, and further characterized as a bone‐selective molecule (MoVattet al., 2002; Thomas et al., 2003). Data suggest expression of this molecule in

developing mineralized tissues, osteoblasts, osteocytes, hypertropic chon-

drocytes, and additionally the PDL region (Bord et al., 2005). Currently,

it is thought that osteocrin may play a role in regulating osteoblast matura-

tion, and subsequently mineral formation. The role of osteocrin in tooth

development, if any, is speculative and requires further investigation.


10. Matrix Gla Protein

Matrix gla protein (MGP), a mineral‐binding extracellular matrix protein,

was originally identified from bone matrix (Price et al., 1983). MGP expres-

sion has subsequently been reported in several types of cells and tissues,

including early in development in lung and limb buds and cells of chondro-

cytic lineage (Luo et al., 1995), and later in mineralized tooth‐associated areas

including dentin and the PDL/cementum region (Camarda et al., 1987; Hale

et al., 1988; Hashimoto et al., 2001).MGPwas also found in tissues producing

unmineralized matrices, cartilage and vascular smooth muscle. MGP has

been proposed to be a negative regulator of mineralized tissues (Mori et al.,

1998), and mice lacking MGP exhibit pathological calcification in arteries,

aortic valves, and cartilage (Luo et al., 1997). MGP expression in the period-

ontium may regulate hard–soft tissue interactions during tooth root develop-

ment, as well as in mature tissues, however, no tooth phenotype has been

found in MGP null mice. It is possible that MGP has a role in root/PDL

development, though its function may overlap that of other mineralization

regulators, for example OPN. Mice deficient in both MGP and OPN had

three times as much arterial calcification by age 4 weeks, and died earlier from

vascular rupture, supporting a shared role of MGP and OPN as inhibitors of

calcification in the vasculature (Speer et al., 2002). A tooth phenotype in

MGP and OPN null mice has not been reported, and it remains to be seen

whether they operate similarly in the periodontal region.

V. Conclusions and Future Directions

In recent years, rapid advances in technologies and molecular biology have

propelled all areas of biomedical research forward at an exciting pace. The

exquisitely regulated, sequential, reciprocal, reiterative cell signaling that

defines morphogenesis and diVerentiation during the development of a tooth

has been well characterized and reported (Jernvall and ThesleV, 2000; ThesleV,2003). Considered together, advances of the last 5–6 years, including increased

understanding of molecular signaling in tooth development, characterization

and employment of tooth stem cell populations, and recombination of cells and

tissues of specific developmental ages to generate tooth structures in vitro, have

truly shown the way to a bright future for tooth engineering.

With this progress in mind, it is now time to delve into how the tooth root

develops and explore the possibilities for regeneration of these tissues, for

the success of bioengineering a ‘‘whole tooth’’ depends on it. Characteriza-

tion of root and associated periodontal apparatus formation lags behind

that of crown: what cells are involved, what signaling drives morphogenesis

Figure 3 Root/PDL formation: key regulators and attractive candidates to consider. While

mechanisms for tooth crown formation have been well established, cells and signals required for

periodontal and tooth root formation are only now beginning to unfold. Naturally occurring

mutations (mut) and knockout (KO) and gene overexpressing (O/E) animal models have driven

the identification of some key regulators, while elucidation of mechanisms controlling root and

periodontal formation is currently underway. During Late Bell stage, root formation is

initiated, preceded by the conversion of the cervical loop to the bilayer Hertwig’s epithelial root

sheath (HERS). A. With proper signaling, the healthy root with associated periodontal

apparatus forms, composed of root dentin (D), cementum (C), periodontal ligament (P), and

alveolar bone (B). B. Alterations in several genes/proteins are known to contribute to cementum

phenotypes, including regulators of Pi/PPi homeostasis (ank, PC‐1, TNAP) and BMP signal-

ing (BMP‐3). C. The development and maintenance of the periodontal ligament (PDL)

is dramatically altered as a result of BMP‐3 overexpression (O/E) and loss of periostin.

D. Alterations on the level of the whole root often lead to tooth loss, including phenotypes

marked by absence of roots (NF1c KO) and distorted roots (Msx2 and DMP‐1 KO, noggin

O/E). Text: Pi ¼ inorganic phosphate; PPi ¼ pyrophosphate; BMP ¼ bone morphogenetic

protein; FGF ¼ fibroblast growth factor; HERS ¼ Hertwig’s epithelial root sheath; NF1c ¼nuclear factor 1c; Shh ¼ sonic hedgehog; KO ¼ knockout; mut ¼ mutation; ank ¼ progressive

ankylosis protein; PC‐1 ¼ plasma cell membrane glycoprotein 1; TNAP ¼ tissue nonspecific

alkaline phosphatase; O/E ¼ overexpressing; PDL ¼ periodontal ligament; DMP‐1 ¼ dentin

matrix protein 1. Insets: IEE ¼ inner enamel epithelium; OEE ¼ outer enamel epithelium;

D ¼ dentin; C ¼ cementum; P ¼ periodontal ligament; B ¼ bone.

102 Foster et al.


and diVerentiation, how is the extracellular matrix synthesized, and how are

cells dynamically aVected by the local environment? This chapter attempted

to pose some of these questions and provide updates regarding current

knowledge on the roles of cells/factors/genes in regulating root and peri-

odontal tissue development, as well as directions for future research. While it

is clear that there is yet much to accomplish, some clues have begun to

emerge regarding tooth root formation. Animal models with periodontal

phenotypes have provided a starting point by indicating transcription fac-

tors, signals and signaling pathways, and matrix proteins that are important

in root formation. Attractive candidates required for controlling root/

periodontal tissue formation, homeostasis, and regeneration have been pre-

sented, and are summarized in Fig. 3. These last years have provided new

insights into the possible triggers for cementum/periodontal tissue develop-

ment and regeneration, and new directions for investigation that should

result ultimately in improved clinical approaches for regeneration of lost

periodontal tissues.

Acknowledgments

Support for this research was provided from grants DE05932 and DE15109 (MJS), and T32

DE0 7023–29 (HKF), from the National Institute of Dental and Craniofacial Research,

National Institutes of Health.

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