Chapter 26
Development of Tooth and Associated
Structures
Eva Matalová,1,2 Vlasta Lungová,3 and Paul Sharpe4
1
Laboratory of Animal Embryology, Institute of Animal Physiology and Genetics CAS, v.v.i., Brno, Czech Republic
2
Department of Physiology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic
3
Faculty of Science, Palacky University, Olomouc, Czech Republic
4
Department of Craniofacial Development and Stem Cell Biology, King’s College London, London, United Kingdom
Chapter Contents
26.1 Odontogenesis 335
26.1.1 Epithelio-Mesenchymal Origin of Dental
Tissues and Embryonic Development 335
26.1.2 Determination of Odontogenic Region and
Tooth Identity 335
26.1.3 Antero-Posterior Patterning 336
26.1.4 Determination of Tooth Shape and Number 336
26.1.5 Morphogenesis 336
26.1.6 Terminal Differentiation and Mineralization 336
26.1.6.1 Dentinogenesis 337
26.1 ODONTOGENESIS
26.1.1  Epithelio-Mesenchymal Origin of
Dental Tissues and Embryonic Development
Dental and periodontal structures are derived from
­swellings of the epithelium lining the developing oral
cavity. Mandibular, nasal, and frontal processes are
mainly populated with the cranial neural crest-derived
ectomesenchymal cells. The cells migrate into different
parts of the developing craniofacial region to eventually
form hard tissues, mechanoreceptors, and melanocytes,
depending on the positional cues from their environment.
Structures of the tooth-alveolar bone complex therefore
develop in context of continuing epithelio-mesenchymal
interactions between the oral ectoderm and the mesenchy-
mal cells of neural crest origin.
Tooth germs start their development prenatally by
specification of an odontogenic field that represents re-
gions of jaw epithelium that have tooth-inducing capacity
and underlying mesenchyme that contains the informa-
tion to specify crown shape (tooth type). Most of the data
Stem Cell Biology and Tissue Engineering in Dental Sciences. http://dx.doi.org/10.1016/B978-0-12-397157-9.00030-8
© 2015 Elsevier Inc. All rights reserved.
26.1.6.2 Amelogenesis 337
26.1.6.3 Cementogenesis 338
26.2 Odontogenesis and Osteogenesis 339
26.2.1 Osteogenesis 339
26.2.2 Alveolar and Jaw Bone Development 339
26.2.3 Alveolar Bone: Extracellular Matrix 342
26.2.4 Root Development and Formation
of Periodontium 342
26.2.5 Failures in Odontogenesis and/or Osteogenesis 344
References 346
on tooth development have been obtained from studies
of the mouse first molar but the findings can be further
extrapolated also for human [1].
26.1.2  Determination of Odontogenic
Region and Tooth Identity
The odontogenic morphogeneic field is believed to be de-
termined by an odontogenic homeobox code [2] by which
spatially restricted domains of homeobox gene expression
in the mesenchyme provide the positional cues. Signals
from the epithelium, in the form of BMP-4 in the distal (in-
cisor) region and FGF-8/9 in the proximal (molar) region,
regulate this spatial expression. Mesenchymal responses
to these signals result in the expression of genes such as
Msx1/2 in incisor regions, and Barx1, Dlx1, 2, 5, and 6 in
molar regions (Figure 26.1).
Inhibition of BMP4, e.g., by its antagonist noggin
or overexpression of Barx-1 in the incisor region, causes
homeotic-like transformation of the incisor tooth germ into
a molar-like tooth [3].
335
336  PART | V  Tooth Tissue Engineering
FIGURE 26.1  Determination of tooth identity. Odontogenic homeobox
code indicates a molecular crosstalk causing formation of incisor tooth
germ in the region of Msx-1 and Msx-2 expression, and molar tooth germ
in the Barx-1 and Dlx-2 expressing region.
Dlx genes also participate in the specification of upper
and lower jaw/teeth identities [4]. Dlx-1 and 2 are sufficient
to form the upper jaw, and Dlx-1, 2, 5, and 6 are required for
formation of the lower jaw. Absence of Dlx-5 and 6 causes
homeotic transformation of the lower jaw into a jaw with
lower jaw identity.
26.1.3  Antero-Posterior Patterning
According to the homeobox code model (Figure 26.1), spa-
tial molecular cues are responsible for the intra- and inter-
jaw patterning set up by expression of Bmp and Fgf genes
in the anterior and posterior oral ectoderm. FGF-8 induces
expression of Lhx-6/7, Dlx-1/2, and Barx-1 (markers of
the posterior oral mesenchyme), and simultaneously elimi-
nates Gsc expression (a marker of the aboral mesenchyme).
BMP-4 present in the anterior oral ectoderm induces ex-
pression of Msx-1/2, and simultaneously restricts Barx-1
expression to the posterior part of the jaw where it contrib-
utes to molar formation. Msx-1 is likely to influence the
growth of the jaw bone, whereas Msx-2 has a stimulatory
effect on alveolar bone growth [5].
26.1.4  Determination of Tooth Shape
and Number
Since persistence of dental lamina is critical for the number
of tooth generations, NF-κB participates in the establish-
ment of tooth numbers within one generation. The resultant
number of teeth, along with the size of the tooth, seems to
be determined early in development by the size of the tooth
field, regulated probably by TNF (tumour necrosis fac-
tor) protein family member Ectodysplasin (Eda). Mutation
in the Eda ligand refers to Tabby, mutation in the recep-
tor EdaR to Downless, and mutation in the adaptor domain
(EdaRadd) to Crinkled phenotype, all causing hypoplastic
hair, teeth, and sweat glands in mutants [6]. Overexpression
of Eda or its ligand initiates formation of an extra tooth with
a premolar-like appearance and position in front of the first
molar tooth.
26.1.5 Morphogenesis
As epithelial budding initiation caused by a shift in orienta-
tion of mitotic spindles in epithelial cells proceeds, the tooth
germ invaginates into the mesenchyme which condenses
around the bud. SHH signaling plays an important role in
regulating the proliferation of the tooth bud epithelium, and
its inhibition causes cell death in the apex of the bud [7].
Later, the epithelium extends farther into the mesenchyme,
and wraps around the condensing mesenchyme forming a
tooth cap-like structure.
As the odontogenic potential shifts to the oral mesen-
chyme, BMP-4 expression shifts to the tooth mesenchyme
and induces formation of the epithelial primary enamel
knot, a signaling center regulating the bud to cap transi-
tion. BMP-4 induces the expression of p21 and Msx-2 in
the underlying epithelium at the tip of the tooth bud, which
is associated with cell cycle arrest, and differentiation of
the primary enamel knot cells secreting mostly mitogenic
molecules. Growth factors (FGFs, SHH) expressed by the
enamel knot stimulate proliferation in adjacent cell com-
partments, but not in the enamel knot itself. Consequent un-
equal proliferation of different subpopulations of the tooth
germ is the main principle of the bud-cap morphogenesis.
In multicuspid teeth, similar signaling clusters of cells (the
secondary enamel knots) control the formation of cusps.
Slit-1 expression in the secondary enamel knot inhibits pro-
liferation, whereas Fgf-4 promotes proliferation of the adja-
cent dental epithelium and mesenchyme, leading to further
epithelial invaginations and cusp formation (Figure 26.2).
Cusp formation probably also involves the Eda signaling
pathway, since mutations in components of this pathway
lead to fewer, shallow cusps, whereas its constitutive activa-
tion leads to molars with many sharp cusps [6].
26.1.6  Terminal Differentiation
and Mineralization
After the basic cusp pattern has been formed, differen-
tiation of dental hard tissue producing cells proceeds.
Mesenchymal cells in contact with the dental epithelium
differentiate into dentin-producing odontoblasts, and the
adjacent layer of inner enamel epithelium cells differenti-
ates into enamel-producing ameloblasts.
Tooth hard tissue formation is therefore governed by
reciprocal interactions between cells of the inner enamel
epithelium and dental pulp lining cells of mesenchymal
origin. Alveolar bone osteoblasts originate from the dental
 Development of Tooth and Associated Structures Chapter | 26   337

FIGURE 26.2  Signalling interactions in tooth development. Initiation covers dental placode formation and epithelial thickening (mouse M1 at E11.5),
and is later followed by bud (E13.5), cup (E14.5), and bell stages. Morphological changes are determined by epithelial (cream) and mesenchymal (red)
molecular signaling, including molecules produced by the enamel knots. PEK: primary enamel knot; SEK: secondary enamel knot; am: ameloblasts; od:
odontoblasts. (Adapted from Thesleff I, J Cell Science 2003, 116, 1647–1648)

mesenchymal cells and allow for functional integration of
the tooth into the jaw bone, together with bone marrow-
derived osteoclasts. Mineralization of the tooth crown
starts at the cusps in the growth centers and continues in
the cervical direction. Formation of the tooth-bone com-
plex hard tissues includes several key processes, particu-
larly dentinogenesis, amelogenesis, cementogenesis, and
osteogenesis [8].
26.1.6.1 Dentinogenesis
Dentinogenesis is the formation of dentin by odonto-
blasts of mesenchymal origin located at the periphery of
the dental pulp (Figure 26.3). Dentinogenesis is initiated
by the inductive influence of the enamel organ involving
molecular signaling pathways, such as Wnt, Runx-2, and
TGF-β. In the molar tooth, dentinogenesis starts at the late
bell stage, and occurs in the crown as well as root regions.
Differentiated odontoblasts stop division and turn into oval
polarized secretory cells with characteristic apical cytoplas-
mic Tomes fibers which connect the cells with the surface
of dentin. Tomes fibers elongate as matrix formation con-
tinues. Predentin, the first organic matrix secreted by odon-
toblasts, is composed by proteoglycans, glycoproteins, and
c­ollagens. Later-formed crystallizing centers of hydroxy-
apatite grow and allow for transformation of predentin into
dentin. Dentinogenesis occurs prenatally as well as postna-
tally, and to a lesser extent can be seen during the whole life
when secondary and tertiary dentin is formed [9,10].
26.1.6.2 Amelogenesis
Amelogenesis is the formation of enamel by ameloblasts of
epithelial origin facing the odontoblast layer (Figure 26.3).
Differentiation of ameloblasts is initiated by more advanced
odontoblasts and the cells of stratum intermedium via mo-
lecular signals, such as BMP and FGF. The major proteins
of the enamel matrix are amelogenins, enamelins, amelo-
blastins, and tuftelins that are produced by the ameloblasts
into the extracellular space. Ameloblastin simultaneously
stops proliferation of ameloblasts, whereas enamelins and
amelogenins are essential for the deposition of hydroxyapa-
tite crystals. Ameloblastins and amelogenins are removed
during maturation when ameloblasts produce proteinases
(e.g., MMP) cleaving these substances. Debris is engulfed
by ameloblasts, and the enamel turns into a highly miner-
alized tissue. Half of the ameloblasts undergo apoptosis
during amelogenesis, the rest die after the process ends.
338  PART | V  Tooth Tissue Engineering

FIGURE 26.3  Initiation of odontogenesis and amelogenesis. BMP and FGF released by odontoblasts induce differentiation of ameloblasts and Wnt,
Runx, and TGF-β produced by ameloblasts in turn induce differentiation of odontoblasts. As differentiation of cells and extracellular matrix deposition
proceeds, a clear dentino-enamel junction forms.

FIGURE 26.4  Distribution of cementum. Acellular cementum is located around the root neck and cellular cementum is covering the root up to the apex.
The cementum is connected with the dentin layer by collagen fibers and with the bony alveolus by Sharpey’s fibers.

Amelogenesis, therefore, stops at the point of tooth erup-
tion, and there is no production of secondary or regenerative
enamel [11,12].
26.1.6.3 Cementogenesis
Cementogenesis is the process of cementum formation
which covers the tooth root by cementoblasts of mesenchy-
mal origin (Figure 26.4). Differentiation of cementoblasts
is not fully understood, but some epithelial components
seem to stimulate dental follicle cells to differentiate into
cementoblasts. Cementoblasts lay down organic matrix
with collagenous fibers where the hydroxyapatite becomes
crystallized and the cementum mineralization proceeds.
The cementum is connected to the dentin layer by colla-
gen fibers, and with the bony alveolus by Sharpey’s fibers.
The cementum occurs as a thinner acellular layer around
the root neck, with thicker cellular cementum covering the
lower part of the root up to the apex, but there is no sharp
boarder between these forms (Figure 26.4). HERS cells are
suggested to secrete acellular cementum in the initial stages
 Development of Tooth and Associated Structures Chapter | 26   339
of cementogenesis. Later on, cellular and reparative cemen-
tum is produced by the dental follicle-derived cementoblast.
Expression studies (DLX-2 and ameloblastin) have sug-
gested different subpopulations of cementoblasts [13,14].
26.2  ODONTOGENESIS AND
OSTEOGENESIS
26.2.1 Osteogenesis
Osteogenesis is a process of bone formation by osteoblasts
of mesenchymal origin, followed by extracellular matrix
mineralization (ossification). Osteogenesis in general occurs
in two major ways—endochondral and intramembranous.
Endochondral ossification is typical in most skeletal
bones, particularly long bones, and is based on pre-existing
cartilage. Endochondral bone formation proceeds in several
steps. First, the pre-skeletal cells migrate from the bone
marrow stroma and start interactions based on the morpho-
genetic gradient. Then condensation and proliferation of
these cells is followed by differentiation and mineralization.
Several molecules are known as essential for individual
steps, as shown in Table 26.1.
Intramembranous (dermal) ossification is character-
ized by direct osteoblast differentiation from mesenchymal
cells without the cartilage step. Intramembranous ossifica-
tion also occurs in the alveolar bone. The bone becomes
formed from condensations of neural-crest derived mes-
enchymal cells that develop into osteoblasts [15]. Unique
features of osteogenic condensations are the requirement
of the epithelium for signaling communication, no Sox-9
expression and early Runx-2 expression. Intramembranous
ossification is triggered in regions where mesenchymal
tissue undergoes a shearing detraction, which originates
from growth of different tissues at different speeds. When
Meckel’s cartilage elongates against its surrounding mes-
enchymal tissue and the outer skin, the adjacent, detracted
mesenchymal cells differentiate into bone forming cells.
TABLE 26.1  Molecules Essential for Individual Steps of Endochondral Ossification
Proliferation and Condensation Proliferation and Differentiation
of Mesenchymal Cells of Chondrocyte Progenitors
TGF-β IGF-1
Wnt 3a, 7a FGF-2/FGFR-2
FGF-2, 4, 8, 10 BMP-2, 4, 7, 14
SHH Sox-9, 5, 6
BMP-2, 4, 7
Sox-9
Gli3
This process starts in the region of the future mental fo-
ramen and similar spatial conditions can be found in the
maxillary region. Remodeling of the bone accompanies
formation of the tooth-bone complex [1].
26.2.2  Alveolar and Jaw Bone Development
Alveolar bone development starts prenatally (at E13 for the
mouse M1) and is based on molecular signaling, as well as
mechanical forces. Two major types of cells participate in
the process—osteoblasts and osteoclasts.
Osteoblasts in the alveolar bone originate directly from
the dental mesenchyme (intramembranous ossification).
After realizing their function in bone matrix production and
mineralization, osteoblasts may undergo programed cell
death, become bone lining cells (inactive osteoblasts), or
become osteocytes, cells encased in the mineralized bone.
Osteoclasts are multinuclear cells that differentiate from
the monocyte-macrophage haematopoietic progenitors re-
cruited from the blood. The differentiation of mononuclear
osteoclast progenitor cells to mature osteoclasts involves
fusion to form multinuclear cells, and their polarization re-
sults in the development of the sealing zone and the ruffled
border required for the attachment to the extracellular bone
matrix and bone resorption.
Several genes participating in odontogenesis can be found
during osteogenesis, particularly Msx-1 and Msx-2, Dlx fam-
ily members (Dlx-1/2, Dlx-5/6), and Runx-2 (Figure 26.5).
Dlx family members regulate skeletal patterning within
the jaw, and in the absence of Dlx-1/2 (downstream of F­ gf-8)
upper molar mesenchyme loses its odontogenic potential
and becomes chondrogenic [16]. Mice deficient in single
Dlx genes or their combinations show various skeletal de-
fects. DLX-5 regulates expression of osteocalcin, a marker
of osteoblasts, RUNX-2 activates expression of collagen
type I, bone sialoprotein, osteocalcin, and osteopontin [17].
Dlx-5, together with Runx-2, also represents differentiation
genes of osteoblasts and osteoclasts (Figure 26.6) [18].
Terminal Differentiation
of Chondrocytes Ossification
FGF-18/FGFR-3 VEGF
BMP-2, 7 FGF-2/FGF-R
Ihh/Ptc Wnt14/β-catenin
Stat 1 Runx2
Gli3, 2 Osterix
Runx2 TCF/Lef1
340  PART | V  Tooth Tissue Engineering

FIGURE 26.5  Illustration of tooth-bone complex development. Formation of alveolar bone is influenced by mechanical pressure and particularly
molecular signaling. Key molecules accompanying integration of the tooth germ with the surrounding bone are shown in the epithelium (cream), mesen-
chyme (red), and bone (yellow).

FIGURE 26.6  Differentiation of osteoblasts. Osteoblasts originate from pluripotent mesenchymal progenitors shared with adipocytes and chondrocytes.
Individual lineages are governed by specific gene expression, Runx2, Dlx5, Msx, and Osx, and key molecules for osteoblast differentiation, Wnt, Runx2,
Dlx5, Mxs, and Osx for following bone mineralization.
 Development of Tooth and Associated Structures Chapter | 26   341

Alveolar bone is missing or abnormally formed in mice
deficient in Runx-2, Dlx-5/6, and Msx-1 genes. Moreover,
application of BMP-4 inhibitors ex vivo causes absence of
alveolar bone formation [19]. Rescue experiments revealed
a network of these genes where MSX-1 seems to act up-
stream of Bmp-4 to activate expression of osteoblast dif-
ferentiation genes Runx-2 and Dlx-5.
BMP family members are critical for bone development,
and in general support bone apposition. In the tooth-bone
complex, bone is the one which has a high capacity for re-
modeling and becomes adapted to the growth of the tooth.
RANK/RANKL/OPG are the best known players in the re-
modeling interplay (Figure 26.7).
After activation of RANK upon binding of its ligand
(RANKL), precursors of osteoclasts undergo differen-
tiation. Whereas increase of OPG, a decoy receptor of
RANKL, causes inhibition of osteoclasts, it supports in-
crease of bone mass leading to delayed tooth development

FIGURE 26.7  Coordination of RANK/RANKL/OPG signaling in osteoclastogenesis. Initiation (left) and inhibition (right) of osteoclastogenesis.
Osteoblasts produce RANKL, osteoclasts have receptors for this ligand (RANK). After RANK-RANKL interaction, osteoclast precursors proliferate,
merge in multicellular structures, and differentiate into matured osteoclasts. Cytokines (and hormones) play important roles in osteoclast differentiation.
Decreased RANKL or increased OPG (decoy receptor) production suppresses osteoclast differentiation.
342  PART | V  Tooth Tissue Engineering
FIGURE 26.8  Coordination of RANKL/BMP2 in tooth eruption. Osteoclastogenesis along the alveolus decreases (decrease of RANKL) whereas, at
the bottom of the alveolus bone, apposition proceeds induced by BMP2 (produced by periodontal cells). The interaction results in bone apposition and
movement of the tooth toward the oral cavity.
and hypomineralization. The RANK/RANKL/OPG team
participates in accommodation of the growing tooth in the
mineralized bone up to eruption, whereas BMP members,
particularly BMP-2, support new bone formation, particu-
larly in the basal part of the alveolus (Figure 26.8) [20].
26.2.3  Alveolar Bone: Extracellular Matrix
Intramembranous bone and dentin are closely related in their
structural features. Seventy percent of bone and dentin are
apatite crystals deposited on a collagen type I scaffold under
the control of noncollagenous proteins secreted by osteo-
blasts and odontoblasts. Differentiation of both odontoblasts
and osteoblasts involves expression of a similar subset of
growth factors, including Wnt, TGF-β superfamily mem-
bers, and Runx-2. Odontoblasts and osteoblasts express a
common subset of extracellular matrix genes: col1a1; dspp;
osteocalcin; bone sialoprotein; and dentin matrix protein-1.
However, the intensity of expression of different extracel-
lular matrix proteins may differ between odontoblasts and
osteoblasts. Dspp is more specific to the odontoblast lineage,
with much lower expression in the bone.
26.2.4  Root Development and Formation
of Periodontium
Histomorphogenesis of the crown region is followed by
histomorphogenesis of the root. The odontogenic epithe-
lium extends apically to form the Hertwig’s epithelial root
sheath (HERS), a bilayer of epithelial cells that induces dif-
ferentiation of dental papilla cells into root odontoblasts.
Once root dentin is formed, HERS loses continuity and the
cells of the inner layer of the dental follicle (mesenchymal
cells surrounding the tooth germ) come into contact with
the root dentin and differentiate into cementoblasts. Cells
of the outer layer of the dental follicle differentiate into fi-
broblasts and osteoblasts that, together with cementoblasts,
contribute to periodontium formation.
Periodontium is a soft tissue at the boarder of the tooth
and bone hard tissues. Periodontium originates from the
mesenchymal cells of the dental sac, and is highly vascular-
ized and innervated. Periodontal tissue consists of heteroge-
nous populations of cells, including fibroblasts, osteoblasts,
cementoblasts, and blood cells, and is rich in collagenous
fibers. Periodontium is a source of stem cells maintaining
homeostasis and allowing for regeneration, and also of sev-
eral signaling factors essential for dental and bone tissue
communication.
Whereas mineralization of enamel, dentin, cementum,
and bone is welcome, mineralization of the periodontium
and dental pulp must be prevented. The prevention is medi-
ated by production of some collagens and secretion of mol-
ecules, such as BMP-3, MSX-2, TWIST, and PLAP [21],
as well as Noggin which suppresses the osteogenic func-
tion of BMP-4 [22].
HERS forms from the cervical loop of the developing
tooth and influences root formation, along with odontoblast
differentiation to secrete root dentin, and cementoblast
differentiation to produce root cementum. Development
of HERS is also based on epithelio-mesenchymal interac-
tions [23], mediated particularly by the interplay of SHH/
MSX-2 and IGF-1/BMP-4 (Figure 26.9). MSX-2, a partner
FIGURE 26.9  HERS and production of signaling molecules. Structure of HERS (left) and fragmentation of HERS (right). BMP4, IGF1, SHH, and
M302 participate in HERS formation and proliferation; BMP2 and 4 induce expression of CP23 and cementogenesis. HERS: Hertwig’s epithelial root
sheath; IEE: inner dental epithelium; OEE: outer dental epithelium.
344  PART | V  Tooth Tissue Engineering
of BMP-4 in crown morphogenesis control, is expressed
in the HERS, and Msx-2 deficient mice have irregularly-
shaped roots [24]. Mutation in the SHH receptor Patched-1
causes repressed proliferation around the HERS, resulting
in disturbed eruption and short roots [25]. Artificial stimu-
lation of IGF-1 receptors results in elongation of HERS and
increased cell proliferation in its outer layer. In the case of
BMP-4 signaling, application of BMP-4 results in short-
ened HERS, whereas the BMP-4 inhibitor Noggin causes
HERS elongation [23]. NFIC, a nuclear factor I family
member, is an essential transcription factor regulating root
odontoblast differentiation. Nfic-deficient mice have abnor-
mal root morphology and lack expression of the odontoblast
marker Dspp [26]. Moreover, HERS cells produce growth
factors, such as BMP-2 and BMP-7 which are able to in-
duce expression of CP23, one of the cementoblast markers
(Figure 26.9) [27]. During root dentin deposition, HERS
becomes fragmented and the remaining tissue is eliminated
by apoptosis, incorporated into cementum, and undergoes
epithelio-mesenchymal transformation, or the cells migrate
to the surrounding periodontium (rests of Malassez). HERS
also seems to establish the root shape during root ­formation.
FIGURE 26.10  Overview of prenatal mouse first mandibular molar development. Distribution of mitosis and apoptosis corresponds with m
changes. ab: alveolar bone; cl: cervical loop; IEE: inner enamel epithelium; oe: oral epithelium; OEE: outer enamel epithelium; mes: mesenchyme.
Surgical damage to HERS correlates with variability in
tooth root growth [28]. For comprehensive overviews of
tooth-bone development see Figures 26.10 and 26.11.
26.2.5  Failures in Odontogenesis
and/or Osteogenesis
As some genes are shared in odontogenetic and osteoge-
netic cascades, failure in their expression and patterning can
affect both structures [1,29]. For example, bone and teeth
are affected in cleidocranial dysplasia (CCD), a congeni-
tal disorder caused by RUNX-2 mutation. Skeletal defects
involve persistently open fontanels, hypoplastic clavicles,
and malformed craniofacial skeleton and vertebrae. The
dental phenotype includes multiple supernumerary teeth
and eruption failure and/or delay, primary dentition failure
to exfoliate, and malocclusion. Mice deficient in the Runx-2
locus completely lack ossification; heterozygous mice show
a similar phenotype to CCD. The most common failure of
the bone with effect on teeth is osteoporosis, a disease when
bone mineral density is decreased and bone microarchitec-
ture disrupted. For example, several studies revealed that
­ orphological
 Development of Tooth and Associated Structures Chapter | 26   345

FIGURE 26.11  Overview of perinatal mouse first mandibular molar development up to eruption. Distribution of mitosis, apoptosis and osteoclast activ-
ity corresponds with morphological changes. ab: alveolar bone; am: ameloblasts; cem: cementum; d: dentin; dp: dental pulp; e: enamel; HERS: Hertwig’s
epithelial root sheath; od: odontoblasts; per: periodontium; SI: stratum intermedium; SR: stellate reticulum; IEE: inner dental epithelium; OEE: outer
dental epithelium.

p­ ostmenopausal women without estrogen substitution ther-
apy have more teeth missing than women with estrogen sub-
stitution therapy. Ovariectomy in mice results in increased
osteoclastogenesis, as well as increased osteoblastogen-
esis, a phenotype observed in postmenopausal women.
Adolescent and adult Opg-deficient mice exhibit increased
activity of osteoclasts and a decrease in total bone density
with a high incidence of fractures. Increased activity of os-
teoclasts also causes familial expansile osteolysis (FEO)
and Paget’s disease. Apart from loss of teeth and dentition
defects, FEO patients suffer from progressive osteoclastic
resorption in long bones followed by fractures, middle ear
defects, and deafness. Paget’s disease displays similar phe-
notypes, and may be caused by mutation in the same gene
as FEO, namely, Rankl, or by mutation in other components
in the RANKL pathway (e.g., sequestome 1). Delayed tooth
eruption, missing or malformed teeth, hypomineraliza-
tion of enamel and dentin, tooth decay, and defects of the
periodontium can occur in the case of osteopetrosis, when
osteoclasts improperly differentiate, and display decreased
activity and function. Osteopetrosis is a complex of dis-
eases ranging from very aggressive lethal forms to milder
phenotypes manifested by bone fractures, frequent infec-
tions, blindness, deafness, and strokes. Up to now, human
346  PART | V  Tooth Tissue Engineering
osteopetrosis was associated with Rankl, Atp6V0A3, Clc7,
grey-lethal homolog, and plecstrin homeodomain contain-
ing protein family M member 1. Osteopetrosis with tooth
eruption defects was observed in mice deficient in genes
critically contributing to osteoclast differentiation and func-
tion: Rankl; Csf1; Pthrp; C-fos; bHLH-Zip; Mitf/Tfe3; and
C-src. Imbalance in calcium and phosphorus homeostasis
of the bone caused, e.g., by mutations in a gene coding tis-
sue nonspecific alkaline phosphatase, may also lead to pre-
mature tooth loss. The opposite case when bone is affected
by tooth-related defects is associated with hypodontia, sur-
gical interventions, trauma, or diseases such as periodon-
titis. Tooth agenesis is in some cases accompanied by loss
or great reduction of alveolar bone, creating problems with
proper fixation of implants. Tooth extraction is followed
by continuous resorption of the remaining alveolar bone
(­alveolar ridge). Chronic inflammation, such as in the case
of periodontitis, is in many cases coupled with excessive
bone resorption, most likely resulting from up-regulation of
RANK/RANKL signaling. Periodontitis may be of a bacte-
rial origin or caused by mutations, such as in the cathepsine
C gene, in the case of Haim-Munk syndrome (HMS) and
Papillon-Lefevre syndrome.
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