21. ODONTOGENESIS - LAMBERT

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ODONTOGENESIS:
RECENT CONCEPTS WITH APPLIED ASPECTS
BY
Dr. Bhuvan Nagpal
B.D.S. (Hons.), M.D.S. (Oral Pathology)
(Gold Medalist)
Consulting Oral & Maxillofacial Pathologist
Ex. Post Graduate Resident,
Dept. of Oral Pathology & Microbiology,
JSS Dental College & Hospital,
JSS University,
Mysuru, Karnataka, India
Dr. Usha Hegde
B.D.S., M.D.S. (Oral Pathology)
Professor & Head,
JSS University,
Dr. Archana S.
B.D.S., M.D.S. (Oral Pathology)
Consulting Oral & Maxillofacial Pathologist
Ex. Post Graduate Resident,
JSS University,

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S.No CONTENTS Page No.
1. INTRODUCTION 3
2. THE EVOLUTION OF DENTITION 6
3.
APPROACHES FOR STUDYING TOOTH
DEVELOPMENT
9
4. DEVELOPMENT OF FACE 13
5. EARLY ODONTOGENESIS 19
6. PATTERNING OF DENTITION 26
7. FACTORS THAT REGULATE TOOTH DEVELOPMENT 34
8. STAGES OF TOOTH DEVELOPMENT 39
9. HISTOPHYSIOLOGY OF TOOTH DEVELOPMENT 69
10. AMELOGENESIS 72
11. DENTINOGENESIS 88
12. DEVELOPMENT OF PULP 101
13. CEMENTOGENESIS 103
14. TOOTH ERUPTION 107
15. SUMMARY 117
16. ANOMALIES OF ODONTOGENESIS 118
17. AGENDA FOR FUTURE 154
18. CONCLUSION 156
19. REFERENCES 158

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INTRODUCTION
“The vertebrate dentition is an evolutionary enigma. It is a critical organ system for
survival and yet it is among the most variable characters in vertebrate history.”1
Odontogenesis deals with the development of various tissues of the teeth (enamel,
dentin, pulp and cementum) and the paradental structures which participate in
anchoring the teeth in its socket (periodontal ligament, alveolar process).1
Teeth are highly mineralized appendages found in the entrance of alimentary canal of
both invertebrates and vertebrates. They are associated mainly with prehension and
processing of food, but they also frequently serve other functions, such as defense,
display of dominance and phonetic alterations in humans.2
Human dentition can be described as diphyodont (two sets of teeth in their life cycle)
and heterodont (different types of teeth such as incisors, canines, premolars and
molars). Moving from the outer to the inner aspect, the part of the tooth (crown) in the
oral cavity is covered by enamel, the hardest tissue in the human body. Inner to the
enamel is the dentin, which is less calcified than enamel but forms the bulk of the
tooth. The dentin surrounds the pulp, which is rich in fibroblast like cells, blood
vessels and nerves. The part of the tooth within the bony socket is covered by
cementum which surrounds the dentin and pulp. The tooth is held in place by the
periodontal ligament.3

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Fig1: Structure of tooth.1
Odontogenesis is a highly co-ordinated and complex process which relies upon cell to
cell interaction that results in the initiation and generation of the tooth. The gross
histological processes are well documented, the mechanisms that are involved at a
molecular level are only now beginning to be elucidated due to the revolution in
molecular biological techniques that has occurred over the last decade.3
During their early development, tooth germs exhibit many morphological and
molecular similarities with other developing epithelial appendages, such as hair
follicles, mammary and salivary glands, lungs, kidneys, etc. The developing tooth

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germ, which is an experimentally accessible model for organogenesis, provides a
powerful tool for elucidating the molecular mechanisms that control the development
of these organs.4
There have been tremendous advances in recent years towards a better understanding
of the regulation of tooth development. The immense interest in this subject is
justified since, apart from the intrinsic scientific merit, congenital abnormalities in
teeth account for 20% of all inherited disorders.2
The variations in the anatomy of the tooth are often over looked upon. The anomalies
that occur during odontogenesis can throw light on a number of underlying conditions
that includes genetic abnormalities, nutritional disturbances, environmental
alterations, infections and many more. Hence, a proper understanding of the various
mechanisms of odontogenesis and their anomalies are of utmost importance to oral
pathologists.2

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THE EVOLUTION OF DENTITION
From an evolutionary-developmental perspective, there are four important features
that make the teeth an attractive model system and are as follows: 5
1. Cusp patterns, tooth shapes and their arrangement in a dental pattern are unique
to a species and is as indicative of a species as its DNA.
2. Because tooth pattern is linked to feeding and hence survival, changes in tooth
pattern provide a major basis for adaptations linked to exploitations of new
feeding niches.
3. Tooth development is a simple process involving only two different cell types.
4. Embryonic tooth development can be easily cultured in vitro to completely
recapitulate normal development.
Simplistically evolution of teeth is believed to have occurred by one of two different
mechanisms:5
1. Teeth evolved independently from jaws from pharyngeal denticles, similar to
those found in many extinct species of fish such as zebra fish.
2. Teeth evolved at the same time as that of jaws by the internalization of skin
denticles (dermal armour) similar to those found on modern day sharks.
Teeth with the basic microscopic anatomy similar to the recent vertebrates first
appeared at ordovicium, approximately 460 million years ago. Some jawless fish
developed superficial dermal structures known as odontodes. These small teeth like
structures were located outside the mouth and served various functions, including

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protection, sensation and hydrodynamic advantage. The encroachment of odontodes
into the oropharyngeal cavity created the buccal teeth, which covered the entire
surface and were later localized to the jaw margins. Dietary habits and ecological
adaptations have driven the teeth of vertebrates to acquire numerous anatomical forms
and shapes, as represented by incisors, canines, premolars and molars.2
Fig 2: Odontodes, the ancestors of teeth looked like placoid scales of recent
sharks. Odontodes consisted of a dentin cone with a pulp cavity and covered by a
hypermineralized tissue like enamel or enameloid. They were attached to the
integument by a bony base.2
Variations in tooth number may represent an important factor for mammalian
diversification. The evolutionary pathway from fish to reptiles to mammals is
characterized by a reduction in the number of teeth (from polyodonty to oligodonty)
and of their generations (polyphyodonty to di/monophyodonty) as well as an increase
in morphological complexity of the teeth (from homodonty to heterodonty).2
Changes in the number and morphology of teeth may reflect a significant factor in the
generation of new species in mammals. The most common feature is the loss of teeth

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as a result of the mutation in tooth related genes. It is worth noting that in placental
mammals teeth tend to disappear over the course of evolution in an order that is
opposite the order of their appearance during eruption. A reaction/ diffusion model of
morphogenesis has been used to explain this phenomenon. According to those models
repeated structures arise as result of co-ordination of two molecules, an activator and
an inhibitor. Teeth located at a distance from the center of the morphogenetic field
tend to disappear due to field attenuation.2
Diet and mastication are regarded as the central factors of teeth evolution. There is a
strong correlation between teeth form and feeding habits. Evolution based on these
aspects allowed for a much more efficient exploitation of the food caloric energy.
Teeth began to evolve from one designed for catching and holding prey to one
designed for better mastication of food. The evolution of mammalian jaw and teeth
created occlusal surfaces that are adequate for a great variety of foods. Triconodont
organisms had three major cusps arranged more or less in a straight line. In
symmetrodont organisms the central cusp was separated from the two outer cusps so
that a triangle was formed on the occlusal surface. With the addition of complimentary
structures the occlusal surface area was dramatically increased leading to an increase
in the masticatory efficiency of molars.2

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APPROACHES FOR STUDYING TOOTH DEVELOPMENT
There are a number of experimental approaches for the study of tooth development.
Of these the three most commonly used methods which have thrown light on the
mechanisms of odontogenesis considerably are:3
1. Genetic analysis of tooth development by means of mouse mutations
2. Organ culture and recombination systems
3. Established cell lines
Genetic analysis of tooth development by means of mouse mutations:
Murine tooth development has proved to be a powerful model to study the genetics
and molecular mechanisms of mammalian tooth development.4
This is mainly
possible because of its suitability for both genetic and embryological manipulation.
Engineered genes can be permanently inserted into the germ line to produce
transgenic mice. Similarly, gene targeting can produce selective gene knockout that
are missing the expression of specific genes.3
It is first useful to distinguish between these two basic techniques. In classic
transgenic technology, a gene is introduced into the mouse germ line by direct
pronuclear injection, under control of either its own promoter or a heterologous
promoter. The normal consequence of preparing a transgenic mouse line which over
expresses or ectopically expresses a particular gene product is a dominant, gain-of-
function mutation which can be transmitted to progeny and its phenotype assessed in
the context of either embryonic or adult development.6

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In knockout technology, a known gene is selectively targeted for disruption in
embryonic stem (ES) cells by the principle of homologous recombination. Following
reconstitution of ES cells in chimeric mice and germ line transmission results in mice
that carry a loss-of-function, typically recessive mutation in a known gene. Two
points regarding this technique deserve special attention. First, a knockout may not
prove informative if the gene whose function is eliminated is required for the
embryonic vitality prior to the time at which developmental process being studied
occurs. Second, many embryonic tissues which normally expresses the tissue may not
exhibit a phenotype.6
Another genetic resource which deserves special mention is the naturally occurring,
spontaneous mouse mutation which exhibit phenotypic defects in tooth development.
These mouse mutations have been helpful only for embryologic and mutational
studies.6
Mouse dentition, however, differs significantly from human dentition. Mice only
develop two different tooth shapes (three molars and one incisor) the region where
humans have canines and premolars. There is a region devoid of teeth in mice called
diastema. Moreover, mice have only one set of teeth while humans have two (one
deciduous and one permanent).4
Organ culture and recombination systems:
It is appreciated for more than 100 years that it is possible to excise tooth rudiments
from rodent embryos and culture them in vitro.6

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1. Experiments involving the use of hanging drop culture methods- the culture
medium is supplemented with chick plasma and chick embryo extract or chick
chorioallantoic membrane, showed that it is possible to grow incisor and molar
tooth germs upto the stage of enamel matrix synthesis.
2. In the presence of 20% horse serum, 10% chick embryo extract and 0.9M
ascorbic acid explanted E17 (bell stage) molar tooth germ could be cultured by
means of trowel-type organ culture system to the stage of odontoblast and
ameloblast differentiation.
3. Serum- free conditions have also been described in which E17 cap-stage tooth
germs can develop to the stage of dentin and enamel matrix synthesis. More
nutritive methods, such as transplantation into the anterior chamber of the
mouse eye, are necessary to permit early tooth germ recombinants to develop
in culture. A refinement of the explants culture technique is the ability to
implant agarose or heparin acrylic beads containing various recombinant
growth factors into isolated dental mesenchyme.
Established cell lines:
A relative paucity of cell culture systems exists for the study of early tooth
development. The major reason for this relates to the difficulties in the
immortalization of early embryonic tissues which retain tooth forming potential. A
series of odontoblastic cell lines has been derived by using a temperature sensitive
large T-antigen-expressing retrovirus vector and mouse odontoblastic cell lines
MDPC-23. Cell lines with dental pulp phenotypes have also been developed. These

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include RDP4-1 and RPC-C2A. Whether these cell lines prove to have a wide utility
for the functional evaluation of potential gene interactions remains to be established.6

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DEVELOPMENT OF FACE
THE STOMATODEUM
The stomatodeum (future mouth) is a depression bounded by a bulges produced by the
brain cranially and by the pericardial cavity caudally. Three prominences appear
around the stomatodeum. These are the frontonasal prominence (above) and the right
and left mandibular arches. The mandibular arch divides into a maxillary and
mandibular process. The right and left mandibular process meet in the midline and
fuse. They form the lower lip and lower jaw. The upper lip is formed by the fusion of
the right and left maxillary process.7
Fig 3: A 27 day old embryo viewed from the front. The beginning elements for
facial development and the boundaries of the stomatodeum are apparent.7

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Fig 4: Human facial development from 24 days through 38 days. Left column
photographs show actual embryos; the middle and right columns are diagrams of
frontal and lateral view.7
A. Boundaries of stomatodeum in a 26-day embryo
B. A 27 day old embryo. The nasal placode about to develop, and the
odontogenic epithelium can be identified
C. 34 day embryo. The nasal pit is surrounded by lateral and medial nasal
process
D. 36 day old embryo shows the fusion of various facial processes that are
completed by 38 days (E)

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Under the light microscope the primitive two or three cell thick layered epithelium
covers the embryonic connective tissue which is termed ectomesenchyme as neural
crest cells have migrated in it. In H&E stained sections, the epithelial cells appear
empty, since the glycogen in these cells is washed outs during tissue preparation.7
The stomodeal ectoderm consits of a basement membrane upon which rests the
cuboidal shaped basal cells. The cells overlying the basal layer are from four to five
cell layers thick and are more rounded. Superficial ones are more squamoid. Electron
microscopy reveals that the cells are connected by desmosomes and the more
superficial cells are poor in organelles.1
The ectomesenchyme consists of a few
spindle-shaped cells seperated by a gelatinous ground substance.7
The role of neural crest cells: As the neural tube forms, the dorsal ectoderm
synthesizes the signaling protein WNT6; whereas in the neural plate, members of the
BNP family are produced. Where these two tissues intersect, active cell multiplication
occurs in both ectoderm and neuroderm. These multiplying cells express FOXD3
gene, which instructs these cells to form two dorsal, longitudinal rows of
ectomesenchyme on both sides of the neural tube to create a transient population of
highly nomadic cells, the neural crest cells.2
The lineage origin of oral ectoderm cells in mammals have not yet been accurately
traced, but the anterior neural ridge rostroventral to the migrating CNC (cranial neural
crest cells) yields the neural epithelium of the head, including olfactory placodes,
rathke’s pouch and the oral epithelium. The ectomesenchyme cells of the developing
facial processes that participate in tooth development form from cranial neural crest

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cells (CNC).8
When the movement of dye injected neural crest cells was traced in
organ cultures of developing dental arches, it was shown that neural crest cells from
the posterior midbrain and to a lesser extent from the anterior midbrain form the
dental ectomesenchyme.8
The failure of neural crest ectomesenchyme to migrate normally to appropriate sites
during craniofacial development leads to serious developmental defects, including the
absence of teeth (anodontia) and underdeveloped jaw bones (micrognathia).8
Fig 5: The source and pattern of neural crest migration to the developing face
and branchial arch system. The midbrain and rhombomere 1 and 2 contribute to
the face and the first branchial arch.7
Lineage and cell analysis has demonstrated a great array of cellular fates arising from
CNC cells, including neurons. Neuroglia, smooth muscle cells, calcitonin producing
C-cells, melanocytes, adipocytes, mesenchymal cells, fibroblasts, cementoblasts,
odontoblasts, chondroblasts, chondrocytes, osteoblasts and osteocytes. The unique

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ability of CNC cells to develop into hard skeletal tissue distinguishes it from trunk
neural crest cells in higher vertebrates, whose cartilage and bone elsewhere in the
body have mesodermal origin.9
Establishment of the oral-aboral axis: To date the earliest mesenchymal markers for
tooth formation are the lim-homeobox domain genes (transcription factors), lhx-6 and
lhx-7.9
The expression of lhx-6 and lhx-7 is restricted to the oral epithelium at sites of
teeth formation, whereas Gsc is expressed posteriorly in the ectomesenchyme that
does not form arch.10
The ectoderm expresses a wide range of signaling molecules,
fibroblast growth factors (Fgfs), Bmps, Wnts and hedgehog proteins (HHs), and it is
the restriction of Fgf8 expression to the oral ectoderm that appears to establish the
antero-posterior axis of the first branchial arch. The restriction of Gsc expression to
aboral mesenchyme involves repression by lhx6/7-expressing cells, although the
mechanism that restricts lhx6/7 expression to oral mesenchyme is independent of Gsc
and is more probably related to the distance from the source of Fgf8. Endothelin 1 is
expressed in the entire mandibular epithelium and appears to act as a maintenance
factor for Gsc gene.9
Targeted mutations of lhx6 or lhx7 do not result in any tooth defects. Such mutations
revealed only when these mutations are combined. Mutations in Gsc, however, do
have a profound mandibular bone phenotype with severe truncation, but teeth develop
normally. Both endothelin1 and endothelin receptor knock-outs have a mandibular
phenotype similar to that of Gsc.9

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The transcription factor Pitx2 defines the oral epithelial area where the teeth will
grow. Deletion of Pitx2 results in complete absence of tooth development before
placode formation. Strong epithelial signals are required for formation of dental
placodes. Several signaling molecules have been implicated as activators (Fgfs, Wnts)
or inhibitors (Bmp) of placode formation. Molecules of ectodysplasin (Eda) are also
implicated in the formation of dentition in mice. Increased Eda signaling in transgenic
mice contributes to larger than normal dental placodes and results in formation of
extra teeth. In contrast inactivation of Eda results in partial tooth agenesis and
misshapen molars. The most sever phenotype is caused by P63 and Runx2 deletion
which causes complete arrest of tooth formation.11

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EARLY ODONTOGENESIS
Initiation of tooth development occurs when the crown-rump length of the embryo is
between 13 and 14mm or about 6.5 weeks of gestation.1
The primary epithelial band
forms a continuous horseshoe shaped sheet of epithelium around the lateral margins of
the developing oral cavity and correspond in position to future dental arches. The
formation of these thickened epithelial bands are a result of not so much of increased
proliferative activity within the epithelium as of change in the orientation of the
mitotic spindle and the cleavage plane of the dividing cell.7
Fig 6: The position of primary epithelial band indicated using shaded areas.7
Fig 7: Change in the plane of cleavage within the dental lamina.7

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The free margin of this band gives rise to two processes, the vestibular lamina and the
dental lamina, which invaginate into the underlying mesenchyme. The outer process,
the vestibular lamina, will form the vestibule that demarcates the cheeks and lips from
the tooth bearing regions. The inner process is the dental lamina and it is from this
dental lamina the tooth buds form.3
The vestibule forms as a result of the proliferation
of the vestibular lamina into the ectomesenchyme. Its cells rapidly enlarge and
degenerate to form a cleft that becomes the vestibule between the cheek and the tooth
bearing areas.7
Continued and localized proliferative activity of the dental lamina leads to the
formation of a series of epithelial outgrowths into the ectomesenchyme at sites
corresponding to the positions of the future deciduous teeth. At this time the mitotic
index, the labeling index and the growth of the epithelium are significantly lower than
corresponding indexes in the underlying ectomesenchyme and ectomesenchymal cells
accumulate around the outgrowths. From this point, tooth development proceeds in
three stages: the bud, cap and bell.7
The most intriguing question that comes to mind at
this juncture is “how is dental development initiated?”
Role of epithelial-mesenchymal interaction: Interactions between epithelial and
mesenchymal tissue components have particularly important function in developing
teeth, as well as in all other organs forming as ectodermal appendages. As shown in
many experimental studies in which the epithelial and mesenchymal tissues have been
recombined and cultured in different heterotypic and heterochronic combinations, the

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interactions are sequential and reciprocal and there is a chain of interaction between
the two tissues driving advancing tooth morphogenesis. 12
When murine first arch epithelium is combined with caudal or cranial neural crest in
the anterior chamber of the eye, teeth forms. Epithelium from other sources does not
elicit this response. However after E12, first arch epithelium loses this odontogenic
potential, which then is assumed by the ectomesenchyme. The ectomesenchyme can
elicit tooth formation from a variety of epithelia. For example at this stage,
recombination of first arch ectomesenchyme with embryonic plantar epithelium
changes the developmental direction of the epithelium so that enamel organ is formed.
Conversely, if epithelial enamel organ is recombined with skin mesenchyme, the
organ loses its dental characteristics and assumes those of epidermis.7
The complicated sequential reciprocal interaction between the dental epithelium and
dental ectomesenchyme that are required for tooth formation are mediated by the
spatiotemporal expression of tooth related genes and the secretion of growth and
transcription factors that are reiteratively used in regulatory loops.2
The bone morphogenetic proteins (BMPs) are homodimeric proteins originally
defined by their ability to induce bone formation invitro. The mammalian BMP family
now consists of eight members, which themselves may be grouped into three
subclasses based upon amino acid similarity. Bmp2, Bmp4 and Bmp7 are all
expressed in the developing molar tooth germ, with Bmp4 and Bmp7 expressed in
both dental epithelium and mesenchyme while, thus far, Bmp2 has been expressed
only in dental epithelium. The expression of Bmp4 begins at E11.5 in the dental

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lamina and shifts at E12.5-13.0 to the dental mesenchyme. This shift in Bmp4
expression coincides temporally with the shift in tooth developmental potential from
epithelium to mesenchyme as deduced from the recombination experiments. Bmp4
can substitute for most but not all the inductive functions of the dental epithelium.
Insitu hybridization experiments indicate that Bmp4 expression is markedly reduced
in the Msx1 mutant dental mesenchyme, while that of other markers, such as the extra
cellular matrix protein tenascin is not.6
Fig 8: Schematic representation of signaling networks mediating sequential and
reciprocal interactions between the dental epithelium and mesenchyme. The
same signaling molecules regulate development at many stages. The genes
indicated in the boxes have been shown to be necessary for the advancement of
tooth morphogenesis in knockout mice. BMP, bone morphogenetic protein; FGF,
fibroblast growth factor; SHH, sonic hedgehog; TNF, tumor necrosis factor.6
In situ hybridization experiments reveal that Msx1 and Msx2 are expressed in the
developing molar tooth germ in, which at least for Msx2, correlate with discrete
morphologic steps in tooth development. Msx2 is first expressed at E10.5 in the

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mesenchyme beneath the site of dental placode formation, and thus constitutes an
early marker for dental initiation. At E11.5, Msx2 is co expressed with Msx1 in the
dental mesenchyme. However, while Msx1 is expressed broadly within the
mandibular mesenchyme in a mesial to distal gradient, Msx2 expression is restricted
to the mesenchyme around the tooth forming regions. Thereafter a component of
Msx2 expression shifts to the epithelium, where it restricts to the enamel knot, while
the mesenchymal domain of Msx2 expression becomes restricted to dental papilla.6
Msx1 is necessary for the mesenchymal expression of Bmp4. Since Bmp4 can induce
the expression of Msx1, it suggests that Msx1 functions to mediate the transfer of
Bmp4 expression from dental epithelium to dental mesenchyme by a positive
feedback loop in the mesenchyme. One possibility of this positive feedback might be
that Msx1 is functioning as a molecular “amplifier”, permitting the more rapid
propagation of Bmp4 inductive signal throughout the dental mesenchyme.6
Expression of Shh is localized to the presumptive dental ectoderm at E11and is thus
another good signaling candidate for tooth initiation. Shh knockout mice have little
development of the facial process and thus any role in tooth initiation cannot be
identified from these. Mutations in Gli genes that are downstream mediators of Shh
action suggest a role in early tooth development because Gli2-/-
and Gli3-/-
double
mutant embryos do not produce any recognizable tooth bud.7
Antagonistic signaling
between Shh and Wnt has been demonstrated to be involved in the definitions of
boundaries of developing tooth germs. Shh expression is restricted to the dental
lamina of the future incisors and molar regions at a very early stage and later confined

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to the tips of the tooth buds. In contrast Wnt7 is expressed throughout the oral
epithelium but is absent in Shh expressing tooth forming regions.13
Lef-1 is a member of the high mobility group family of nuclear proteins that includes
the t-cell factor proteins, known to be nuclear mediators of Wnt signaling. Lef 1 is
first expressed in dental epithelial thickenings and during bud formation shifts to
being expressed in dental epithelia thickenings and during bud formation shifts to
being expressed in the condensing mesenchyme. In Lef-1 knockout mice, all dental
development is arrested in bud stage. Ectopic expression Lef-1 in the oral epithelium
results in ectopic tooth formation.7
Expression of several genes in ectomesenchyme marks the sites of tooth germ
initiation. These include Pax-9 and Activin-A, both of which are expressed around
E11 in mice within localized groups of cells corresponding to where tooth epithelium
will invaginate to form buds. In the case of Pax-9, antagonistic interaction between
Fgf-8 and Bmp-4, possibly act to regulate Pax-9 expression. Pax9 has an important
role in regulation of cellular pluripotency and differentiation during embryonic
patterning and organogenesis.10
Activin-A expression which is not regulated by the
same mechanism, suggesting that Fgf-8 and Bmp-4 interaction may not have a direct
role in tooth initiation.7

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Fig 9: Schematic representation of the signals and transcription factors
mediating the reciprocal signaling between epithelium and mesenchyme during
advancing tooth development.7

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PATTERNING OF DENTITION
The determination of specific tooth types at their correct positions in the jaws is
referred to as patterning of the dentition. The determination of crown process is a
remarkably consistent process. Although in some animals teeth are all of the same
shape (homodont), in most mammals they are different (heterodont), falling into three
families: incisiform, caniniform and molariform. The patterning is tightly controlled:
transpositions are occasionally seen, but they usually involve teeth at the border of a
particular series (i.e. canines and premolars) and more severe anomalies of patterning
(i.e. molars developing at the front of the arch) do not occur.7
Classically two theories have been proposed to account for this.
The field theory (Butler, 1939): This theory suggests that all tooth primordia are
initially equivalent, with the individual shape that they subsequently develop into
being controlled by varying concentration of morphogens in the local environment. A
number of diffusible signaling molecules have been identified that may be involved in
concentration-dependant, threshold response mechanisms which would produce
periodicity along the developing dental axis. However, if these mechanisms are
responsible for patterning in both dentition, then they must act very early on in the
development process. Unlike the mandibular dental axis the developing maxillary
dentition is not continuous. The maxillary incisors develop in the medial nasal
processes, whilst the remainder of the dentition develops in the maxillary process of
the first arch.3

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Clonal model (Osborn, 1978): In this model, the tooth primordial are said to be
prespecified with each migrating cell population being equipped with the necessary
positional information to produce different classes of teeth from inception. Migration
of the neural crest cells from the region of the developing hindbrain provides much of
the mesenchyme of the developing orofacial region; including that contributing to
odontogenesis.3
Histological data of discrete initiation favor the clone model rather
than the field model of a diffusible morphogen. However, Westgaard and Ferguson
have proposed a hybrid ‘progress zone model’ where the progressive disto-proximal
restriction of Hox-8 expression in epithelium and mesenchyme coincides with this
model.7
Fig 10: Clone theory:10
A) The molar clone ectomesenchyme has induced the dental lamina to begin
tooth development. The clone and dental lamina progress posteriorly.
B) When a clone reaches the critical size, a tooth bud is initiated at its centre,
C) The next tooth bud is not initiated unless the progress zone of the clone
escapes the influence of a zone of inhibition surrounding the tooth bud

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The homeobox code model for dental patterning is based on observations of the
spatially restricted expression of several homeobox genes in the jaw primordial
ectomesenchyme cells before E11. The early expression of Msx-1 and Msx-2
homeobox genes before the initiation of tooth germs is restricted to distal, midline
ectomesenchyme in regions where incisors and canines but not multicuspid teeth
develop; whereas Dlx-1 and Dlx-2 are expressed in ectomesenchyme cells where
multicuspid teeth, but not incisors or canines develop. These expression domains are
broad and do not exactly correspond to specific tooth types. Rather they are
considered to define broad territories.7
Expression of Barx-1 overlaps with Dlx-1 and Dlx-2 and corresponds closely to
ectomesenchyaml cells that develop into molars. The homeobox code model proposes
that the overlapping domains of these genes provide the positional information for
tooth type determination. Support for this model comes from the dental phenotype of
Dlx-1-/-
and Dlx-2-/-
double knockout mice in which development of maxillary molar
teeth is arrested in epithelial thickening stage. As predicted by this model, incisor
development is normal in these mice. Further support for this model comes from
misexpression of Barx-1 in distal ectomesenchyme cells, which results in incisor tooth
germs developing as molars. FGF-8 in proximal ectoderm induces Barx-1 expression
whereas Bmp-4 in the distal ectoderm represses Barx-1 expression. Experimentally
induced expression of barx-1 in distal ectomesenchyme by inhibition of BMP
signaling has the effect of repressing Msx gene expression, which is induced in distal
mesenchyme by BMP-4.7

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There are three different conclusions from this model. The first is that there is no one
specific gene for each tooth type. Second, the code is both positive and negative; thus
the absence of a gene is as important as its presence. Third, the code is overlapping
and can thus provide morphogenetic cues for many different tooth shapes.9
Fig 11: Migrating neural crest cells express the same homeobox (Hox) genes as
their precursors in the rhombomeres from which they derive. Note that Hox
genes are not expressed anterior to rhombomere 3. A new set of patterning genes
(Msx, Dlx, Barx) has evolved to bring about development of cephalic structures
so that a “Hox code” also is transferred to the brachial arches and developing
face.7

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Fig 12: Homeobox code model for dental patterning.7
A. Domains of Barx-1 and Dlx-1/-2 expression overlap in the mesenchyme of
the presumptive molar region, whereas domains of Msx-1, Msx-2 and Alx-
3 overlap in presumptive incisor mesenchyme.
B. Mouse dental pattern. Incisors deriving from MSX-1/Alx-3 expressing
cells, molars deriving from Barx-1/Dlx-1/ Dlx-2 expressing cells
C. Human dental pattern. Premolars and canines can be derived from the
same odontogenic code as that observed in mice by virtue of the
overlapping domains of gene expression. Thus canines and premolars may
be derived from cells expressing Dlx-1/-2 and Msx-1, for example.

31
An obvious question therefore is how are highly restricted domains of
ectomesenchymal gene expression regulated? Two possible mechanisms are that: (1)
neural crest cells contain a pre pattern and (2) neural crest cells respond to positional
signals from the oral epithelium. Removal of epithelium from mandibular arches at
E10 or before, results in a total and rapid loss of almost all ectomesenchymal
homeobox gene expression. Removal of epithelium at E10.5 also results in loss of
gene expression and subsequent addition of FGF8 beads restores expression in the
original expression domains only. Removal of epithelium at E11 does not affect gene
expression, indicating that the spatial homeobox expression domains are established
and maintained in the absence of epithelial signals.14
Fig 13: Schematic representation explains the signaling interdependence between
epithelium (E) and ectomesenchyme (EM).14
A. The uncommitted mesenchyme cells equally responsive and dependand on
epithelium for signals.
B. The domains of ectomesenchymal gene expression become fixed but still
dependant on epithelium for signals.
C. The fixed gene expression domains of the ectomesenchyme are no more
dependent on epithelium.

32
Up to E10, all ectomesenchyme cells appear to be uncommitted and competent to
respond to epithelial signals regardless of position. By E10.5, the spatial expression
domains have been established in the ectomesenchyme by the action of epithelial
signals such as FGF8 and BMP4. By E11, expression of spatial ectomesenchymal
genes does not require epithelial signals. Epithelial signals thus regulate the spatial
expression of homeobox genes in the ectomesenchyme which in turn control
morphogenetic pathways, probably by influencing enamel knot function. The control
of tooth shape thus mirrors the general control of tooth formation, with information
being passed from epithelium to ectomesenchyme and back again to epithelium.8
Recombination experiments have revealed much important information about the
rostrocaudal positioning of tooth and arch patterning.
1. Primarily it is that the first brachial arch epithelium is unique in containing
instructive signals for odontogenesis and these can over-ride the pre-patterning
information present in the neural crest cells.
2. The maxillary and mandibular epithelia are interchangeable as regulators of
ectomesenchymal gene expression. If this is true, then the instructive signals
must produce identical differentiation pathways which are not the case, as it is
obvious that different skeletal structures and subtly different teeth are produced
in spite of being covered by same epithelium.14
Functional redundancy and their complexities: Despite both the genes being
expressed in identical patterns on the proximal maxilla and mandibular primordia, the
normal development of mandibular molars and the failure of maxillary molars to

33
develop in Dlx1/2 double mutants indicate a basic genetic difference between the
specification of molar morphogenesis during development of upper and lower jaws.
Dlx5 and Dlx6 are co-expressed in proximal ectomesenchyme of mandibular
primordial in domains similar to Dlx1 and Dlx2. Significantly however Dlx5 and Dlx6
are not expressed in the maxillary arch. Mutations in Dlx1 and/or DLx2 affect maxilla
development, presumably because Dlx5 and Dlx6 genes carry out this function in the
mandible in the absence of Dlx1 and Dlx2.8
The activin enigma: Activin is a member of the TGFβ family of growth factors.
Activin proteins function as dimmers consisting of βA and βB subunits encoded
respectively by activin βA and activin βB genes. Activin βA expression is localized in
presumptive tooth mesenchyme of all teeth, where it acts as an early mesenchymal to
epithelial signal. Surprisingly mouse mutants for activin βA lack all teeth except the
maxillary molars. This phenotype is reciprocal of Dlx1/2 phenotype. The Dlx1/2
phenotype can be explained by functional redundancy with other Dlx genes, whereas
the activin βA phenotype cannot be explained by redundancy, since activin βA/βB
double mutants have the same phenotype as activin βA single mutants.4
The most obvious explanation for the development of maxillary molars in the absence
of activin is that the role of activin in these teeth is carried out by another TGFβ
family ligand, binding to activin receptor and stimulating the same pathway. This
seems not to be the case, since the expression of activin signaling target genes, such as
follistatin, is lost in the maxillary molar tooth germs in activin βA mutants. The
molecular basis of this phenotype is yet to be explained.8

34
FACTORS THAT REGULATE TOOTH DEVELOPMENT
Vitamin A and its metabolic derivatives, retinol and retinoic acid (RA), are essential
regulators of epithelial cell proliferation and differentiation and have special impact
on tooth development. The importance of vitamin A was underscored by the
observation that when endogenous vitamin A was blocked in vitro, dental lamina fails
to develop in organ cultures of mouse embryonic mandible. Early studies on the effect
of vitamin A on tooth development showed that a deficiency of it leads to defective
enamel and dentin. In contrast, excessive vitamin A increases the chance of tooth bud
fusion and/or the formation of supernumerary teeth.8
In organ cultures of embryonic mandibular explants, retinol and retinoic acid increase
epithelial proliferation and stimulate the formation of extra tooth buds. Retinoic acid
exerts its effect by binding to nuclear transcription factors (RA receptors [RARs])
located near retinoid response elements on various target genes, one being the gene
that produces the epidermal growth factor. Retinoic acid also increases the expression
of midkine (MK) protein, a regulator of cell proliferation.8
Cellular retinol binding protein (CRBPs) and cellular retinoic acid-binding protein
(CRABPs) are involved in the metabolism and storage of vitamin A metabolites in the
cytoplasm. Both RARs and CRABPs have been localized in the dental lamina and
adjacent ectomesenchyme as well as in the dental epithelium and ectomesenchymal
components of developing teeth. In addition, CRABPs have been localized in the
epithelium adjacent to the sites of dental lamina formation, suggesting that RA may be
bound at such sites. In the dental lamina, where there appears to be fewer CRABPs,

35
the RA molecules are free to interact with their nuclear receptors and thereby increase
the expression of EGF.8
Fig 14: Cellular action, retinoic acid (RA) the major metabolite, of vitamin A
diffuses into the cell interior, where it binds to cellular retinoic acid-binding
protein (CRABP), or, if the level of CRABP is low, may enter the nucleus to
interact with its receptor (RAR). Retinoic acid receptors activate retinoic acid
response elements that regulate gene transcription, thereby stimulating the
production of mRNA. The epidermal growth factor gene is regulated by RAR-
RARE complex. The increase in cell proliferation effected by vitamin A is
believed to be result of secretion of epidermal growth factor (EGF), a known
mitogen for dental epithelium and ectomesenchyme.8
Epidermal growth factor, acting in a paracrine or autocrine manner, appears to control
the rate of cell proliferation in the early stages of tooth development. Epithelial cells
of the dental lamina and early enamel organ expresses EGF receptor. When the
enamel organ reaches the cap stage of development the level of binding of EGF
decreases in the epithelial cells but increases in the ectomesenchymal cells of the
underlying dental papilla. Interfering with the synthesis of EGF blocks
odontogenesis.8

36
Another RA regulated gene expressed during tooth development is midkine (MK).
The MK gene and its product are preferentially located in the embryonic tissues
undergoing epithelial mesenchymal interaction. Both MK mRNA and MK protein are
preferentially expressed in all stages of developing maxillary and mandibular teeth of
embryonic mice. The differential or appositional localization of MK mRNA and MK
protein in developing dental ectomesenchyme and its receptor on the cells of the IEE
provides an instructive example of epithelial mesenchymal interaction. During cap
stage of tooth development, the MK protein is secreted by the ectomesenchymal cells
and concentrated in the basal lamina. The significance of MK in tooth development is
confirmed by observation that antibodies to MK inhibit odontogenesis. The highest
level of MK is observed in the IEE, its basal lamina, the dental papilla and especially
in the differentiating odontoblasts. With the onset of dentin secretion, MK is no longer
detectable in odontoblasts or in the differentiating preameloblasts.8
Fig 15: Appositional pattern of the expression of midkine (MK) gene in ectomesenchyme and
the localization of the MK protein (MKp) to the surface of the inner enamel epithelial cells
adjacent to the basement membrane (BM) of a cap stage tooth bud. The diffusible MK protein
is concentrated in the BM and is bound to cell surface receptors (MK-R) on epithelial cells,
where it may act as a paracrine signaling molecule. Although EM cells make MK protein they
lack receptors IEE- inner enamel epithelium, DP-dental papilla EO-enamel organ.8

37
Neurotrophins and neurotrophin receptors are expressed in developing teeth in
association with preameloblasts and preodontoblasts. They are also expressed in the
sub odontoblastic layer. Neurotrophins play a central role in the development and
maintenance of nerves. Recent studies suggest that neurotrophins are expressed early
in dental epithelium before the developing teeth are innervated. The presence of
neurotrophins and receptors in developing teeth, and their changing spatiotemporal
relations suggest, in addition to a role in dental neuronal development, they may have
other non neuronal regulatory functions.8
Nerve growth factor is a ligand for the tyrosine kinase receptor A, member of the
neurotrophin receptor family. Nerve growth factor produced in the developing tooth
may act locally to control the number of cell cycles in the IEE and the dental papilla
proliferation compartments. The expression of nerve growth factor receptor decreases
as the cell division in the IEE ceases prior to ameloblast differentiation.8
Growth hormone, growth hormone-binding protein, and growth hormone receptor
have been localized in the developing teeth. Cells of the enamel organ and dental
papilla appear to be the targets for growth hormone. Likewise, insulin like growth
factor is concentrated in the IEE and dental papilla during ameloblast and odontoblast
differentiation. Hepatocyte growth factor and its receptor are expressed in the dental
papilla. Hepatocyte growth factor acts as a mitogen in regulating cell proliferation in
enamel organ and dental papilla. Antisense nucleotides to hepatocyte growth factor
reduce mitotic activity in the IEE and dental papilla, leading to abnormal tooth
development.8

38
The neurotransmitter serotonin (5-hydroxytryptamine) is another potential
morphogenetic signaling molecule. Specific uptake of serotonin occurs transiently in
the oral epithelium and developing teeth. Tooth buds grown in the presence of
inhibitors to serotonin uptake fail to develop beyond the bud stage.8

39
STAGES OF TOOTH DEVELOPMENT
At certain points along the dental lamina, each representing the location of one of the
10 mandibular and 10 maxillary deciduous teeth, the ectodermal cells multiply still
more rapidly and form little knobs that grow into the underlying mesenchyme. Each
of these down growth from the dental lamina represents the beginning of the enamel
organ of the tooth bud of a deciduous tooth. Not all of these enamel organs start to
develop at the same time, and the first to appear are those of the anterior mandibular
region.14
As the cell proliferation continues, each enamel organ increases in size, sinks deeper
into the ectomesenchyme (dental papilla) and due to differential growth changes its
shape. As it develops it takes on the shape that resembles a cap, with the outer convex
surface facing the oral cavity and an inner concavity.14
The shape of the enamel organ continues to change. The depression occupied by the
dental papilla deepens until the enamel organ assumes a shape resembling a bell. As
the development takes place the dental lamina, which had thus far connected the
enamel organ to the oral epithelium, becomes longer and thinner and finally breaks up
and the tooth bud loses its connection with the epithelium.14
Although tooth development is a continuous process, the developmental history of a
tooth is divided into several morphologic stages for descriptive purposes. While the
size and shape of the individual teeth are different, they pass through similar stages of

40
development. They are named after the shape of the enamel organ and are called the
bud, cap and bell stages.14
Fig 16: Diagrammatic representation of the life cycle of tooth.14

41
Stages in tooth growth14
Morphologic stages Physiologic process
Dental lamina Initiation
Bud stage
Cap stage Proliferation
Bell stage (early) Histodifferentiation
Bell stage (advanced) Morphodifferentiation
Formation of enamel
And dentin matrix Apposition
THE DENTAL LAMINAE
In the first stage of tooth development increased mitotic activity in a specific portion
of the stomodeal ectoderm of both arches produces a prominent thickening which
dips into the adjacent mesenchyme. Epithelial proliferation progresses bilaterally
eventually forming two horseshoe shaped bands defining the prospective upper and
lower dental arches. The germinal band of ectodermal epithelium circumscribing the
future maxillary and mandibular arches is the dental lamina.1
The cell components of the dental lamina are neither cytologically nor
morphologically similar along its length. Electron microscopic studies of human
odontogenesis indicate that dissimilarities in the cells exist to the extent that four
zones may be recognized. These are the orodental epithelial junction, area of epithelial

42
rests formation, the intermediate cord and the free terminal. The tip of the dental
lamina is composed of a bulbous compact cell mass. It is separated from the adjacent
connective tissue by a basement membrane which follows the topography of the
external cell surface. The connective tissue surrounding the tip is sheath like in its
arrangement.1
Fig 17: Primary epithelial band at the sixth week of intra-uterine life. (H&E;
X115)14
The external cells of the tip are cuboidal to low columnar in shape with few
intercellular spaces. Their adjacent epithelial cells are rounder and less compactly
arranged. The organelle population in the tip cells is more numerous than those of the
other areas of the dental lamina. The tissue surrounding the intermediate cord, though
in intimate association does not exhibit the sheath like arrangement to the extent on
that of the tip. The cord cells differ from those of the tip in that their profiles, in
general are more irregular. The region above the cord is the longest and is
characterized by the formation of cell aggregations as cords or islands surrounded by a

43
basal lamina and connective tissue. Components of this region of the dental lamina
not engaged in the formation of cell rests are stellate with irregular intercellular
spaces. The outer cells of the orodental epithelial junction rest on a basement
membrane which is continuous with that of the future oral epithelium on one side and
the area of cell rest formation on the other.1
Fig 18: The vestibular lamina (A) and dental lamina (B) at the seventh week of
intra-uterine development. (H&E, X120)14
Vestibular lamina: The vestibular laminae grows into the ectomesenchyme as a
broad band of epithelium, separating the connective tissue mass into two territories, a
larger one associated with the lips and cheeks and a smaller one with the upper and
lower arches. The unusual characteristic of the vestibular lamina is that as the sheet
thickens, the central most cells separate to form a cleft, the oral vestibule. Of the two
approximating epithelial sheets, the external one forms the lining of the labial and
buccal mucosa, and the internal one forms the gingival epithelium covering the
vestibular aspect of the arches. Thus the vestibular lamina effects the separation of the
stomodeal mass into the lips or cheek and the developing arches. The vestibular

44
lamina is also known by names such as the lip furrow band, labial or buccal lamina,
buccogingival lamina and vestibular lamina.1
Fig 19: Stages of developing dental lamina (DL) and vestibular lamina (VL). A,
The tongue (T) is separated from the adjacent tissue mass by a sulcus (LS).
Dental lamina grows into underlying mesenchyme (Me). B, dental lamina
continues its downward migration into the mesenchyme and the vestibular
lamina (VL) is initiated. Note that the dental arch and cheek form a solid mass.
C, dental lamina has progressed deeply into mesenchyme and its distal terminal
forms a bulbous mass (bud). Vestibular lamina invades mesenchyme more deeply
and its central cells undergo autolysis forms a cleft which marks the beginning of
the vestibule (V). D, distal terminal of dental lamina (DL) forms a cap shaped
dental organ primordium (TG). The lip (L) and dental arch (D) are delineated by
the cavitation or vestibule (V) of the vestibular lamina. Lingual sulcus (LS)1

45
Fig 20: The developing dental lamina. (Masson’s trichrome, X55)1
Successional laminae: The portion of the dental lamina adjacent to the developing
tooth anlage retains its connection with the lingual aspect of the tooth primordium via
the lateral lamina. The free terminal of the dental lamina begins to proliferate in the
fourth month of fetal growth (55-100nm). This newly established growth center is
known as the successional (succedaneous) lamina and is destined to provide the
anlage for permanent teeth replacing the primary predecessors. While the 20 dental
primordial for the primary dentition are established within a week, their replacements
require an excess of a year.1
Parent dental laminae: In the seventh week of development, tooth anlagen for 20
primary teeth are formed by the dental lamina. This lamina also provides tooth germs
for the permanent teeth which have no primary predecessors. Because of this, the
dental laminae providing for the formation of the first, second, and third permanent
molars may be referred to as the parent dental laminae or the laminae for permanent
molars. The mechanism involved is simply one of continued distal growth. That is, the
distal ends of the dental laminae for each arch, after having established the tooth
germs for the primary molars, continues to grow posteriorly. These segments of the

46
dental laminae elongate progressively, keeping pace with the lengthening of the
arches.1
The buds for the permanent first molar appear in the embryo at four months in utero;
the others are produced after birth. The buds for the second molars appear in the infant
of 9 months and those of the third molars about the age of four years. Thus, activity of
the various dental laminae begins about 6.5 weeks of embryonic development and
continues postnatally to the age of four years. After the establishment of the
primordial for permanent molars.1
Rudimentary laminae: Once the tooth germs for the deciduous dentition have
become established and progress toward the appositional stage, the epithelial cord
representing the dental lamina exhibits signs of disorganization. Disorganization of
dental lamina is initiated first at the orodental epithelial junction and progress toward
the deeper core. Epithelial remnants of the rudiments of dental laminae were once
known as the Glands of Serres because of the glandular configuration of the epithelial
groups.1
Fig 21: Epithelial pearls of Serres.1

47
THE BUD STAGE
The second stage of odontogenesis is called the bud stage and occurs at the beginning
of the eighth week of prenatal development for primary dentition. This stage is named
for an extensive proliferation, or growth, of dental lamina into buds or oval masses
penetrating onto the ectomesenchyme. At the ends of the proliferation process
involving the primary dentition’s dental lamina, both the future maxillary arch and the
future mandibular arch will each have ten buds.15
The components of the bud are a compactly arranged mass of similar cells. That is,
except for the core, the cell components are morphologically and cytologically
similar. Cell surfacing the bud and hence the mesenchyme are low columnar or
cuboidal in shape. While the basal lamina over most of the bud conforms faithfully to
the contour of the cell bases, such is not the case for the cells on the superior surface.
The core cells range in shape from round to stellate with prominent intercellular
spaces.1
The cells of the tooth bud have a higher RNA content than those of the
overlying oral epithelium, a lower glycogen content and increased oxidative enzyme
activity.16
Electron microscopic studies of these cells reveal that the organelle populations,
relative to their lack of maturity, are extensive. Perinuclear accumulations of the
tonofilaments are especially prominent. An ectoplasmic zone is present in most cell
components of the bud, but is especially wide in cell bordering areas in which the
basal lamina does not follow the contour of the cell bases. These features, width of
ectoplasmic layer and non conformance of basal lamina path, tend to suggest that they

48
are associated with cell movements and specifically with reorganization of the cells of
the primordia in the transformation from bud to the cap stage.1
Fig 22: Epithelial invagination into ectomesenchyme.14
As a result of the increased mitotic activity and the migration of neural crest cells into
the area, the ectomesenchymal cells surrounding the tooth bud condense. The area of
ectomesenchymal condensation immediately subjacent to the enamel organ is the
dental papilla. The condensed ectomesenchyme that surrounds the tooth bud and the
dental papilla is the dental sac. Both the dental papilla and the dental sac become well
defined as the enamel organ grows into the cap and bell stages.14
Fig 23: Tooth bud formation.14

49
The question now arises as to how the ectomesenchymal condensation takes place. To
date fibronectin, fibronectin receptors, tenascin and syndecan have been implicated as
responsible for the condensation of ectomesenchyme.8
Syndecan, a proteoglycan cell adhesion molecule located in the cell membrane is
expressed prior to tooth formation in the ectomesenchymal cells that underlie the
dental epithelium. Tenascin a large substrate adhesion molecule is expressed in the
ectomesenchyme during the down growth of the dental lamina and during subsequent
condensation of the dental papilla. It has been proposed that the binding of the
membrane bound syndecan molecules to extracellular tenascin molecule is responsible
for the condensation of the ectomesenchymal cells.8
An alternative explanation is that tenascin interferes with cell to fibronectin
attachment, leading to decreased migration of ectomesenchymal cells causing them to
aggregate in the form of dental papilla. Adhesion of fibroblast is weaker to fibronectin
than to tenascin. It has also been shown that when cells express syndecan they have a
reduced ability to invade a collagen gel. Thus, the appearance on syndecan on the cell
surface of ectomesenchymal cells may have a direct, negative effect on their ability to
migrate, thereby causing them to form aggregates, such as the dental papilla.8
Genetic aspects of tooth bud formation: Dental placodes secrete molecules from all
four growth and transcription factor families (BMPs, FGFs SHH and WNTS) which
induce the expression of many genes (PAX9, MSX1/2, RUNX2, BMPs,
FGF.ACTIVIN, LEF1) in the mesenchyme. Specifically, epithelial BMP4 induces the
production of mesenchymal BMP4, whereas epithelial FGF8 induces mesenchymal

50
activin βA. BMPs and FGFs activate MSX1, whereas FGFs induce the expression of
PAX9 and RUNX2. The epithelial cells under the influence of BMP4 and activin βA,
start proliferating and intrude within the mesenchyme in a cylinder like structure with
a bulb like bud at the end. The expression of PAX9 is responsible for mesenchymal
condensation. During the bud stage of tooth development, the odontogenic potential is
lost from the epithelium (around ED 11.5-12) and gained by the ectomesenchyme.2
Bud to cap transition: The transition from bud to cap marks the onset of
morphologic differences between tooth germs that give rise to different types of teeth.
Msx-1 is expressed with Bmp-4 in the mesenchymal cells that condense around tooth
buds. MSx-1-/-
embryos have tooth development arrested at the bud stage, and Bmp-4
expression is lost from mesenchyme, suggesting that Msx-1 is required for Bmp-4
expression.7
Bmp-4 expression in the bud mesenchyme is required to maintain Bmp-2 and Shh
expression in the epithelium. Loss of Bmp-4 expression in Msx-1 mutants is
accompanied by loss of Shh expression at E12.5, which can be restored by exogenous
Bmp-4. Blocking SHH signaling using neutralizing antibodies shows that at E11-E12
Shh is required for dental epithelium proliferation to form tooth buds, whereas
blocking at E13 affects tooth bud morphology, but these buds can still form teeth.7
Another homeobox gene involved in bud to cap transition is Pax-9. Pax-9 is expressed
in bud stage mesenchyme and also earlier in domains similar to activin βA and Msx-1
in patches of mesenchyme that mark the sites of tooth formation. Pax-9-/-
mutants have

51
all teeth arrested in the bud stage. Thus, Pax-9 and Activin βA is essential for tooth
formation beyond the bud stage and appear to function independently.7
THE CAP STAGE
The third stage of odontogenesis is called the cap stage and occurs for the primary
dentition between the ninth and tenth week of prenatal development. The physiologic
process of proliferation continues during this stage, but the tooth bud of the dental
lamina does not grow into a large sphere surrounded by ectomesenchyme. Instead,
there is unequal growth in different parts of the tooth bud, leading to the formation of
a cap shape attached to the dental lamina.15
Thus not only does proliferation characterize this stage, but various levels of
differentiation (cytodifferentiation, histodifferentiation and morphodifferentiation) are
also active during the cap stage. Additionally during this stage, a primordium of the
tooth develops with a specific form. Therefore the predominant physiological process
during the cap stage is one of morphogenesis.15
Fig 24: Cap stage of tooth formation.15

52
As the tooth bud grows larger, it drags along with it part of the dental lamina; so from
that point on, the developing tooth is tethered to the dental lamina by an extension
called the lateral lamina. At this early stage of tooth development, identifying the
formative elements of tooth is already possible.7
The epithelial outgrowth, which superficially resembles a cap sitting on a ball of
condensed ectomesenchyme is referred to widely as the dental organ but actually
called the enamel organ, because it eventually forms the enamel of the tooth. The
enamel niche is an apparent structure in histologic sections, created because the dental
lamina is a sheet rather than a single strand and often contains a concavity filled with
connective tissue. A section through this arrangement creates the impression that the
tooth germ has a double attachment to the oral epithelium by two separate strands.7
Fig 25: Enamel niche.7
The ball of condensed ectomesenchymal cells, called the dental papilla, will form the
dentin and the pulp. The condensed ectomesenchyme limiting the dental papilla and

53
encapsulating the enamel organ, the dental follicle or sac, gives rise to supporting
tissues of the tooth.7
Early in the ontogeny of the tooth, those structures giving rise to the dental tissues
(enamel, dentin, pulp and supporting apparatus) can be identified as discrete entities.
Important developmental changes begin late in the cap stage and continue during the
transition of the tooth germ from cap to bell. Through these changes, termed
histodifferentiation, a mass of similar epithelial cells transforms itself into
morphologically and functionally distinct components.7
Fig 26: Cap stage of tooth development.7
The cells in the center of the enamel organ synthesize and secrete glycosaminoglycans
into the extracellular compartments between the epithelial cells. Glycosaminoglycans
are hydrophilic and hence pull water into the enamel organ. The increasing amount of
fluid increases the volume of extracellular compartment of the enamel organ, and the

54
central cells are forced apart. Because they retain connections with each other through
their desmosomal contacts, they become star shaped. The center of the enamel organ
is thus termed the stellate reticulum.7
The role of enamel knot: The enamel knot is composed of a transient population of
non dividing epithelial cells that appear during the late bud stage of development at
the site of primary tooth cusps. The enamel knot precursor cells can be detected first at
the tip of the tooth buds by expression of the p21 gene, followed shortly after by Shh.
By the cap stage when the enamel knot is visible histologically, it expresses genes for
many signaling molecules including Bmp-2, Bmp-4, Fgf-4, Wnt-10b, Slit-1 and Shh.
Three-dimensional expression of these genes have revealed highly dynamic spatial
and temporal nested patterns in the enamel knot as it extends between the inner and
outer enamel epithelia as the enamel cord.7
It is thought that the enamel knot acts as a signaling center, being responsible for
directing cell proliferation and subsequent cuspal morphogenesis in the developing
enamel organ. In the molar teeth the secondary enamel knot also appears at sites of
the future secondary cusps, almost certainly under the influence of the primary enamel
knot. Both the primary and the secondary enamel knots express Fgf-4 and are non-
dividing; Fgf-4 is known to stimulate the proliferation of both epithelium and
mesenchyme. It has been proposed that this induced cell proliferation of the enamel
organ in conjunction with the lack of cell division in the enamel knot allows the
growth and the folding of the developing cusps. At the cap stage of tooth
development, the cells of the enamel knot undergo apoptosis and disappear,

55
presumably switching off its signaling function. The enamel knot is formed during the
late bud stage of tooth formation when the capacity to induce tooth morphogenesis is
known to reside in the mesenchyme.3
The proposed signaling function of the enamel knot implies that an epithelial derived
structure does have a regulatory role to play in the later stages of odontogenesis. The
enamel knot is seemingly necessary for morphogenesis of the tooth germ to progress
from bud to the cap stage.3
Fig 27: The enamel knot.7

56
Fig 28: Localization of Fgf-4 mRNA (red stain) in a cap stage lower molar tooth
by insitu hybridization. Intense expression can be seen in the enamel knot.3
THE BELL STAGE
The fourth stage of odontogenesis is the bell stage. which occurs for the primary
dentition between the eleventh and twelfth week of prenatal development. It is
characterized by continuation of the ongoing process of proliferation, differentiation
and morphogenesis. However, differentiation on all levels occurs to its furthest extent,
and as a result, four different types of cells are now found within the enamel organ.
These cell types form layers and include the inner enamel epithelium, the outer
enamel epithelium, the stellate reticulum, and stratum intermedium. Thus the cap
shape of enamel organ evident in the last stage assumes a bell shape.15
During this stage, the tooth crown assumes its final shape (morphodifferentiation), and
the cells that will be making the hard tissues of the crown (ameloblast and
odontoblasts) acquire their distinctive phenotype (histodifferentiation).7

57
The bell stage can be divided into:
x Early bell stage
x Late bell stage
Early bell stage: The configuration of internal enamel epithelium broadly maps out
the occlusal pattern of the crown of the tooth. This folding is related to differential
mitosis along the inner enamel epithelium. The future cusps and incisal margins are
sites of precocious cell maturation associated with cessation of mitosis, while areas
corresponding to the fissures and margins of the tooth remain mitotically active. Thus,
cusp height is related to continued downward growth at the margin and fissures than
to upward extension of the cusps. During the bell stage, any bone resorption defects
that restrict the space for development of tooth germ may be associated with increased
folding pattern of the internal enamel epithelium, leading to changes in tooth shape.16
Fig 29: Early bell stage of tooth development. The undersurface of the enamel
organ has deepened, giving the organ its bell shape. The dental papilla and dental
follicle are evident.16

58
Fig 30: Early bell stage of tooth development A-inner investing layer of dental
follicle, B- outer layer of dental follicle. (Masson’s trichrome, X45).16
It is during the bell stage of development that the dental lamia breaks down and the
enamel organ loses connection with the oral epithelium. At the same time the dental
lamina between tooth germs also degenerates. Remnants of the dental lamina may
remain in the adult mucosa as clumps of resting cells (epithelial pearls of serres) that
may contain keratin and may be involved in the etiology of cysts.16
Interposed between the enamel organ and the wall of the developing bone crypt is the
mesenchymal tissue of the dental follicle, which is generally considered to have three
layers. The inner investing layer is vascular showing fibrocellular condensation, three
to four cell thickness, immediately surrounding the tooth germ; the nuclei of the cells
tend to be elongated circumferentially. The outer layer of dental follicle is represented
by a vascular mesenchymal layer that lines the developing alveolus. Between the two
layers is loose connective tissue with no marked concentration of blood vessels. There

59
is evidence that the cells of the inner layer of the dental follicle may be derived from
the neural crest.16
A high degree of histodifferentiation is achieved in early bell stage. The enamel organ
shows four distinct layers: external enamel epithelium, stellate reticulum, stratum
intermedium and internal enamel epithelium.16
Except for the stellate reticulum which
occupies the core and bulk of the dental organ and which is designated as such
because of the reticulum arrangement of its stellate shaped components, the other
three layers are named so because of their locations. The name of the outer enamel
epithelium is given because it forms the outer surface of the bell, the inner enamel
epithelium because it forms the inner lining of the bell, and the stratum intermedium
because it is located intermediate to the stellate reticulum and inner enamel
epithelium.1
The cervical loop at the margin of the enlarging bell shaped enamel organ is a site of
mitotic activity. Here, the central cells of the stellate reticulum/stratum intermedium
may be the site of stem cell niche providing cells that pass to the inner enamel
epithelium and forms ameloblasts. This may be under the control of notch proteins in
the epithelium and growth factors, such as BMP4 and FGF10, in the adjacent dental
mesenchyme.14
Inner enamel epithelium: The inner enamel epithelium consists of a single layer of
cells that differentiate prior to amelogenesis into tall columnar cells called
ameloblasts. These cells are 4 to 5μm in diameter and about 40μm high. The cells of

60
the inner enamel epithelium exert an organizational influence on the underlying
mesenchymal cells in the dental papilla, which later differentiate into odontoblasts.14
Stratum intermedium: A few layers of squamous cells form the stratum intermedium,
between the inner enamel epithelium and the stellate reticulum. The well developed
cytoplasmic organelles, acid mucopolysaccharides and glycogen deposits indicate a
high degree of metabolic activity. This layer seems to be essential to enamel
formation. It is absent in the part of the tooth germ that outlines the root portion of the
tooth which does not form the enamel.14
Stellate reticulum: When compared with that of the cap stage, the stellate reticulum in
the bell stage expands further, mainly by an increase in the amount of intercellular
fluid. The cells are star shaped, with long processes that anastomose with those of
adjacent cells. Before the enamel formation begins, the stellate reticulum collapses,
reducing the distance between the centrally situated ameloblasts and the nutrient
capillaries near the outer enamel epithelium. Its cells are then hardly distinguishable
from those of the stratum intermedium. This change begins at the height of the cusp or
the incisal edge and progresses cervically.14
Outer enamel epithelium: As the name suggests, this forms the outer layer of
cuboidal cells that limits the enamel organ. The external enamel epithelium is thought
to be involved in the maintenance of the shape of the enamel organ and in the
exchange of substances between the enamel organ and the environment. The cervical
loop at which there is considerable mitotic activity, lies at the growing margin of the

61
enamel organ where the outer enamel epithelium is continuous with the inner enamel
epithelium.16
Late bell stage: The late bell stage of tooth development is associated with the
formation of dental hard tissues, commencing at about the 18th
week. Dentine
formation always precedes enamel formation. Down growths of the external enamel
epithelium appears from the lingual sides of the enamel organs. In deciduous teeth,
these lingual down growths give rise to the tooth germs of the permanent successors
and first appear alongside the incisors at about 5 months in utero. In enamel organs of
permanent teeth, however, these down growths eventually disappear. Behind the
deciduous second molar the dental lamina grows backwards to bud off successively
the permanent molar teeth. The first permanent molar teeth appears about 6 months in
utero, the tooth buds of the second permanent molar appears about 6 months after
birth, while that of the third permanent molar appears at about 4-5 years after birth.
Under the inductive influence of developing ameloblasts (pre-ameloblasts), the
adjacent mesenchymal cells of the dental papilla become columnar and differentiate
into odontoblasts. The odontoblasts then become involved in the formation of
predentine and dentin. The presence of dentin then induces the ameloblasts to secrete
enamel.16
ROOT DEVELOPMENT
Root development is initiated through the contributions of the cells originating from
the enamel organ, dental papilla and dental follicle. The cells of the outer enamel
epithelium and inner enamel epithelium contact at the base of the enamel organ, the

62
cervical loop. Later as the crown is completed the cells of the cervical loop continue to
grow away from the crown and become the root sheath cells. The inner root sheath
cells cause root formation by inducing the cells of the dental papilla to form
odontoblasts, which in turn will form root dentin. The root sheath will dictate whether
the root will be single or multiple. The remainder of the cells of the dental papilla will
form the pulp. The cells of the dental follicle form the supporting structures of the
teeth, the cementum and the periodontal ligament.17
Root sheath development: After the crown is completed the inner and outer enamel
epithelium at the base of the cervical loop proliferates to form a bilayer of epithelial
cells called the Hertwig’s epithelial root sheath. The first formed part of the epithelial
root sheath bends upward at a 450
angle to form a disc like structure, the epithelial
diaphragm. It reduces the size of the primary apical opening, which finally becomes
the apical foramen. The epithelial diaphragm maintains a constant size during root
development because the continuity of the root sheath grows in length at the angle of
the diaphragm and not at its tip. With increased root length the crown begins to grow
away from the base of the crypt. This uplifting of the tooth provides space needed for
continued tooth growth. As a result the epithelial diaphragm maintains its position in
relation to the base of the crypt. The root therefore lengthens at the same rate as the
tooth erupts.17

63
Fig 31: Hertwig’s epithelial root sheath.17
Fig 32: Beginning of root development.17

64
Fig 33: Diagram showing three stages of root formation (A) Section through the
tooth germ, note epithelial diaphragm and proliferation zone of pulp (B) higher
magnification of cervical region of A (C) imaginary stage showing the elongation
of hertwig’s epithelial root sheath coronal to diaphragm. Differentiation of
odontoblast in elongated pulp. (D) In an area of proliferation, dentin has formed.
Root sheath is broken into epithelial rests and is separated from the dentin by
connective tissue.17
Single root formation: Formation of the single rooted sheath occurs through the
growth of the root sheath, like a cuff or tube, around the cells of the dental pulp
followed by development of root dentin. Cells of the inner layer of root sheath induce

65
the adjacent cells of the dental papilla to differentiate into odontoblasts, which in turn
form dentin. As the first layer of dentinal matrix mineralizes, the epithelial root sheath
cells separate from the surface of the root dentin and breaks occur in its continuity.
The separated root sheath cells then begin to migrate away from the root surface
deeper into the follicular areas. Mesenchymal or ectomesenchymal cells of the dental
follicle then migrate between the remaining epithelial cell groups to contact the root
surface. At this surface they differentiate into cementoblast and secrete cemental
matrix (cementoid) which subsequently mineralizes to form cementum. Root
elongation continues progressively, with proliferation of the remaining root sheath
cells at the base of the angle of the epithelial diaphragm. As the root lengthens the
compensatory movement of eruption provides space for further root development.17
Multi root formation: Human multirooted teeth have in common a root trunk, which
is the area of the common root base located between the cervical enamel and the area
between which the root division occurs. Development of the multirooted teeth
proceeds in much of the same way as that of the single rooted teeth until the furcation
zone is completed. Division of the root takes place by the differential growth of the
root sheath. In the region of the epithelial diaphragm, tongue like extensions develop
and grow until contact is made with one or two opposing extensions that fuse with
each other. This divides the original single opening of the root trunk into two or three
openings. The epithelium then continues to proliferate at an equal rate at the perimeter
of each of the openings and forms epithelial diaphragms and cuffs to map the
individual root to map the individual roots as they elongate. The area of contacts of

66
the tongue-like extensions forms epithelial bridges at the furcation zone. At each
bridge, the inner cells of the epithelial root sheath induce formation of odontoblasts,
which in turn will produce a ‘span’ of dentin between and around each root.
Odontoblasts then continue to proliferate along the coronal pulpal floor. Dentin
formation will then follow the root sheath and produce the multiple roots.17
Fig 34: Stages in development of two rooted tooth diagrammatic mesio-distal
sections of a lower molar (A) Beginning of dentin formation at bifurcation (B)
Formation of two roots in progress.17

67
Fig 35: Three stages in the development of tooth with two roots and three roots.
Surface view of epithelial diaphragm. (A) Expands eccentrically so that
horizontal epithelial flaps are formed later these horizontal flaps proliferate and
unite (dotted lines in C) and divide single cervical opening into two or three
openings.17
Fate of Hertwig’s epithelial root sheath: After dentin formation the epithelial root
sheath breaks down, and its remnants migrate away from the dentinal surface. These
remnants come to lie some distance from the root in the periodontal ligament, and
become the epithelial rests of Malassez. These cells persist in the periodontal ligament
throughout life. They are often found near the apical zone in young individuals up to
20 years of age. Later these cells tend to be seen more in the cervical areas of the

68
tooth. This is because the epithelial cells have an inherent characteristic of moving to
the surface and exfoliating.17
Microscopically, epithelial cells appear either as a network of epithelial strands along
the root surface, as isolated islands of cells surrounded by connective tissue, or as
isolated cells in close contact with the cementum, three types of epithelial cells
develop proliferating, resting and degenerating. This description is dependent on
whether the cells are dividing, inactive, or undergoing cell lysis.17
Fig 36: Epithelial cell rests of Malassez.17

69
HISTOPHYSIOLOGY OF TOOTH DEVELOPMENT
A number of physiologic growth processes participate in the progressive development
of the teeth. Except for their initiation which is a momentary event, these processes
overlap considerably, and many are continuous throughout the various morphologic
stages of odontogenesis. Nevertheless, each physiologic process tends to predominate
in one stage more than the other.14
The five physiologic stages of tooth development are:
1. Initiation
2. Proliferation
3. Histodifferentiation
4. Morphodifferentiation
5. Apposition
Initiation: The dental laminae and associated tooth buds represent those parts of the
oral epithelium that have the potential for tooth formation. Different teeth are initiated
at different times. Initiation induction requires ectomesenchymal-epithelial
interaction. It has been demonstrated that dental papilla mesenchyme can induce or
instruct tooth epithelium and even non tooth epithelium to form enamel.14
Proliferation: Enhanced proliferative activity ensues at the points of initiation and
results successively in the bud, cap and bell stages of the odontogenic organ.
Proliferative growth causes a regular change in the size and proportion of the growing
tooth germ. Even during the stage of proliferation, the tooth germ already has the

70
potential to become more highly developed. This is illustrated by the fact that explants
of these early stages continue to develop in tissue culture through the subsequent
stages of histodifferentiation and appositional growth.14
Histodifferentiation: The formative cells of the tooth germs developing during
proliferative stage undergo definite morphologic and functional changes and acquire
their functional assignment. The cells differentiate and give up their capacity to
multiply as they assume their new function; this law governs all differentiating cells.
This phase reaches its highest development in the bell stage of the enamel organ, just
preceding the beginning of formation and apposition of dentin and enamel. The cells
of the inner enamel epithelium causes the differentiation of the cells of the dental
papilla into odontoblasts during the bell stage. With the formation of dentin the cells
of the inner enamel epithelium differentiate into ameloblasts and enamel matrix is
formed opposite the dentin. Dentin formation therefore precedes and is essential for
enamel formation.14
Morphodifferentiation: The morphologic pattern, or basic form and relative size of
the future tooth, is established in morphodifferentiation, that is, by differential growth.
Morphodifferentiation is therefore impossible without proliferation. The advanced bell
stage marks not only active histodifferentiation but also an important stage of
morphodifferentiation in the crown, outlining the future dentinoenamel junction.
The dentinoenamel and dentinocemental junctions, which are different and
characteristic for each type of tooth, act as a blue print pattern. In conformity with this
pattern the ameloblast, odontoblast and cementoblast deposit enamel dentin and

71
cementum respectively and thus give the completed tooth its characteristic form and
size.14
Apposition: Apposition is the deposition of the matrix of the hard dental structures.
Appositional growth of the enamel and dentin is a layer like deposition of an
extracellular matrix. This type of growth is therefore additive. It is the fulfillment of
the plans outlined at the stages of histodifferentiation and morphodifferentiation.
Appositional growth is characterized by regular and rhythmic deposition of the
extracellular matrix, which is of itself incapable of further growth. Periods of activity
and rest alternate at definite intervals during tooth formation.14

72
AMELOGENESIS
Amelogenesis, or enamel formation is a two step process. When enamel first forms, it
mineralizes only partially to approximately 30%. Subsequently as the organic matrix
breaks down and greater than 90% organic matrix is removed, crystals grow wider
and thicker. This process whereby organic matrix and water are lost and mineral is
added accentuates after the full thickness of the enamel layer has been formed to attain
greater than 96% mineral content.7
Regulation of ameloblast differentiation: The differentiation of ameloblast and
odontoblast are regulated by epithelial mesenchymal interactions, like tooth
morphogenesis and the same signaling molecules have been implicated. TGFβ
superfamily signals regulate both enamel and dentin formation. Recent evidence from
transgeneic mice indicates that BMP4 is the major signaling molecule regulating
ameloblast differentiation and enamel formation. This study also revealed an
inhibitory function for the dental follicle in amelogenesis. In has been shown that
activin from dental follicle induces follistatin expression in preameloblasts, and that
follistatin in turn antagonizes the function of odontoblast-derived Bmp4 as an
ameloblast inducer.12
Light microscopy of amelogenesis: At the late bell stage, most of the light
microscopic features of amelogenesis can be seen in single section. Thus in the region
of the cervical loop the low columnar cells of the inner enamel epithelium are clearly
identifiable. As the inner enamel epithelium is traced coronally in a crown stage tooth
germ, its cells become tall and columnar, and the nuclei become aligned at the

73
proximal ends of the cells adjacent to the stratum intermedium. Shortly after dentin
formation initiates, a number of distinct and almost simultaneous morphologic
changes associated with the onset of amelogenesis occur in the enamel organ. The
cells in the IEE, now ameloblasts begin more actively to secrete enamel proteins that
accumulate and immediately participate in the formation of a partially mineralized
initial layer of enamel which does not contain any rods. As the first layer of enamel is
formed, ameloblast move away from the dentin surface. Enamel is identified readily
as a deep staining layer in demineralized hematoxylin-eosin sections. An important
process in the production and organization of enamel is the development of
cytoplasmic extension of ameloblasts, Tomes’ process, that juts into and interdigitates
with the newly forming enamel. In the sections of forming human teeth, tomes process
give the junction between the enamel and ameloblast a picket fence or saw tooth
appearance.7
When the formation of full thickness of enamel is complete, ameloblasts enter the
maturation stage typically this stage starts with a brief transitional phase during which
significant morphologic changes occur. These post secretory transitional ameloblasts
shorten themselves and restructure themselves onto squatter maturation cells.7
Cells from the underlying stratum intermedium, stellate reticulum, and outer enamel
epithelium reorganize so that recognizing individual layers is no longer possible.
Blood vessels invaginate deeply into cells without disrupting the basal lamina
associated with the outer aspect of the enamel organ to form a convoluted structure
referred to as the papillary layer.7

74
Finally, when enamel is fully mature, the ameloblast layer and the adjacent papillary
layer regress and together constitute the reduced enamel epithelium. The ameloblasts
stop modulating, reduce the size and assume a cuboidal appearance. This epithelium
although no longer involved in the secretion and maturation of enamel, continues to
cover it and has a protective function. In case of premature break of the epithelium,
connective tissue cells are believed to come into contact with the enamel and deposit
cementum on the enamel. During this protective phase however the composition of
enamel can still be modified. The reduced enamel epithelium remains until the tooth
erupts. As the tooth passes through the oral epithelium, the part of the reduced enamel
epithelium situated incisaly is destroyed, whereas that found cervically interacts with
the oral epithelium to form the junctional epithelium.7
Fig 37: Light microscopy of enamel formation7

75
ELECTRON MICROSCOPY OF AMELOGENESIS
Ultrastructural studies of enamel formation by electron microscopy have added greatly
to the understanding of this complex process. Amelogenesis has been described in as
many as six phases but is generally subdivided into three main functional stages
referred to as the presecretory, secretory and maturation stages. Classically,
ameloblasts from each stage have been portrayed as filling more or less exclusive
functions.7
Fig 38: The various functional stages of ameloblast as would occur in a human
tooth. (1) Morphogenetic stage; (2) Histodifferentiation stage (3) initial secretory
stage (no tomes’ process); (5) ruffle ended ameloblast of the maturative stage; (6)
smooth ended ameloblast of the maturation stage; (7) protective stage.7

76
Presecretory stage:
Morphogenetic stage: during the bell stage of tooth development, the shape of the
crown is determined. A basal lamina is present between the outer enamel epithelium
and the dental follicle and between the cells of the inner enamel epithelium and dental
papilla. The cells of the inner enamel epithelium still can undergo mitotic division.
They are cuboidal or low columnar, with a large centrally located nucleus and poorly
developed golgi elements in the proximal portion of the cell7
Differentiation phase: As the cells of the inner enamel epithelium differentiate into
ameloblasts, they elongate and their nuclei shift proximally towards the stratum
intermedium. The basal lamina supporting them is fragmented by cytoplasmic
projections and disintegrates during mantle predentin formation. The golgi complex
increases in volume and migrates distally from its proximal position to occupy a major
portion of the supranuclear cytoplasm. The endoplasmic reticulum and mitochondria
increases and clusters significantly in the proximal region. A second junctional
complex develops at the distal extremity of the cell, compartmentalizing the
ameloblast into a body and a distal extension called the tomes process against which
the enamel forms. Thus the ameloblast becomes a polarized cell, with the majority of
the organelles situated in the cell body distal to the nucleus. These cells can no longer
divide.7
Although in the past these differentiating ameloblasts have been regarded as non
secreting cells, research now clearly demonstrates that production of some enamel
proteins starts much earlier than anticipated, even before the basal lamina separating

77
the preameloblast and the preodontoblasts is lost. Adjacent ameloblasts are aligned
closely with each other, and attachment specializations, or junctional complexes,
between them maintain the alignment. These complexes encircle the cell at their distal
and proximal extremities. These junctional complexes play an important role in
amelogenesis by tightly holding together ameloblasts and determining at different
times what may, and what may not, pass between them to enter or leave enamel.7
Secretory stage: At the beginning of the secretory phase, the ameloblast have become
long, columnar cells over 60μm in height and 2-4μm in width, with their nuclei at the
basal end. Following the deposition of initial thin aprismatic enamel, a cone shaped
process, tomes process, forms at the distal, secretory end of the ameloblasts. The
shape of the tomes process is responsible for the prismatic structure of enamel. There
appears to be a relationship between ameloblast size and prism pattern. It is usually
found that pattern 3 prisms are made by the largest ameloblasts and pattern 2 by the
smallest. With the development of the tomes process the shape of the mineralizing
front changes to a “picket fence” arrangement.16
Fig 39: TEM showing advanced secretory ameloblasts with their Tomes process.
B developing enamel.16

78
Fig 40: Section showing secretory ameloblasts, (en) enamel. It is to be noted that
the nucleus is placed away from the forming enamel.16
As the ameloblasts shift from the presecretory to the secretory stage, there is a marked
aggregation of vesicles at the distal end of ameloblasts. The material contained within
the vesicles represents the organic matrix of enamel. The contents of the enamel are
discharged into the extracellular space, both at the distal end of the cell and between
the cell membranes of adjacent ameloblasts. As the enamel matrix is secreted the
ameloblasts are pushed outwards away from the dentin surface. Within the organic
matrix, the initial hydroxyapatite crystallites of the enamel appear almost
immediately. The first formed crystallites are thin and needle like and much smaller
than the crystallites in mature enamel.16
Enamel prisms elongate incrementally. Each daily increment leads to cross striations.
Approximately every 7 days prominent cross striations produce the appearance of
enamel striae. These striae end in the surface called perikymata. In teeth that are

79
mineralizing at birth there is an exaggerated incremental line, the neonatal line. The
secretory phase ends once the full thickness of the enamel matrix has been laid down.
The tomes process retracts so that the distal ends become flat and a final thin layer of
aprismatic enamel is formed on the surface. The crystallites in the surface enamel all
run parallel to each other.16
Maturation stage: Before the tooth erupts in the oral cavity, enamel hardens. Crystal
growth during the maturation stage occurs at the expense of matrix proteins and
enamel fluid that are largely absent from the mature enamel. Although the maturation
stage ameloblasts generally are considered to as post secretory cells, they still
synthesize and secrete proteins. Theses ameloblasts still exhibit a prominent golgi
complex, a structural feature consistent with such activity. The significance of
continued matrix production while major matrix removal occurs is unclear.7
Transitional phase: Ameloblasts now undergo significant morphologic changes in
preparation for their next functional role, that of maturing the enamel. A brief
transitional phase involving a reduction in height of ameloblasts and a decrease in
their volume and organelle content occurs. During the maturation stage the
ameloblasts undergo programmed cell death.7
Maturation proper: Next the principal activity of ameloblast is the bulk removal of
water and organic material from the enamel to allow introduction of additional
inorganic material. The most visually dramatic activity of these cells is modulation,
the cyclic creation, loss and recreation of a highly invaginated ruffled border or a
smooth border. Modulation can be visualized by special stains and occurs in waves

80
travelling across the crown of a developing tooth from least mature regions to most
mature regions of the enamel.7
Fig 41: Functional morphology of ruffle ended and smooth ended maturation
stage ameloblasts.7
The significance of modulation is uncertain, but they seem to be related to
maintaining an environment that allows accretion of mineral content and loss of
organic matrix, in part through alteration in the permeability of enamel organ. Ruffle
ended ameloblasts possess proximal junctions that are leaky and distal junctions that
are tight, whereas most smooth ended ameloblasts have distal junctions that are leaky
and proximal junctions that are tight.7

81
Fig 42: Model showing the proposed intracellular movement of calcium in
ameloblasts.7
Data available suggest that the calcium ions required for active crystal growth pass
through the ruffle ended ameloblast (because their distal junctions are tight) but along
the sides of the more leaky smooth ended ameloblasts. Active incorporation of
mineral ions into crystals occurs in relation to ruffle-ended ameloblasts. Regarding the
withdrawal of organic matrix from maturing enamel, sufficient evidence now exists to
indicate that active resorption of intact proteins by ameloblasts is not the main
mechanism for the loss of organic matrix observed during enamel formation. This is
attributed largely to the action of bulk-degrading enzymes that act extracellularly to
digest the various matrix proteins into fragments small enough to be able to leave the
enamel layer. Polypeptide fragment leaving the enamel likely pass between the leaky
distal junctions of smooth ended cells and diffuse laterally among the ameloblasts to

82
be taken up along their basolateral surface. Just as ameloblasts complete the
transitional phase and begin the first series of modulation cycles, they deposit a basal
lamina at their now flattened apex. The basal lamina adheres to the enamel surface
and the ameloblasts attach to it by means of hemidesmosomes. Typical basal lamina
constituents such as collagen type IV have not been demonstrated. However the basal
lamina has been shown to contain laminin-5, which is essential for the formation of
hemidesmosomes. Patients with laminin-5 deficiency show focal enamel hypoplasia.
Also the basal lamina is situated such that it could relay to the ameloblast information
about the status of the dynamic enamel component.7
Fig 43: The relationship between tomes process and enamel prism formation.
The enamel of the core of the prism boundary/interred region differ largely in
the orientation of crystals this is determined by the shape of the tomes process.
Each prism is formed by single ameloblast but four contribute to each
interprismatic region. The prism boundary areas are formed first, giving the
developing enamel a pit like configuration.7
Protective stage: As the enamel maturation nears completion, the ameloblast now
secrete a material between the now flattened distal ends of the cells and the enamel

83
surface. This material appears morphologically identical to the basal laminin. The
ameloblasts at this stage protect the newly formed enamel surface from the follicular
connective tissue. If they fail for whatever reason the connective tissue cells
differentiate into cementoblasts and deposit cementum on the enamel surface. During
this protective phase however the cell is still able to modify the enamel composition.
For instance fluoride if available can still be incorporated into the enamel of unerupted
teeth, and there is evidence that fluoride content is greatest in those teeth that have the
longest interregnum between the completion of enamel formation and tooth eruption.7
Uniqueness of amelogenesis: Amelogenesis is unique in many ways. The secretory
cell is an epithelial cell whereas all other secretory cells of hard tissues are
ectomesenchymal. Non-collagenous hard tissues are involved in mineralization of
enamel whereas in all other hard tissues collagen plays an important role. The matrix
of enamel does not contain collagen whereas in other hard tissues collagen is the
major protein. The matrix of enamel is partially mineralized whereas in other tissues it
is non mineralized. Enamel therefore lacks a distinct organic phase such as osteoid,
predentin or cementoid. There is no absorption of secreted matrix in other hard tissues
but in enamel formation 90% of secreted matrix is absorbed and this activity is done
by ameloblasts itself. After enamel formation ameloblasts undergo apoptosis hence
enamel formation does not occur later on.14

21. ODONTOGENESIS - LAMBERT

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