1. Tooth Development - I Man-Kyo Chung, DMD, PhD (mchung@umaryland.edu)
Assistant Professor
Department of Neural and Pain Sciences
University of Maryland Dental School

2. Developmental histology of tooth
zygote
(a cell from fertilization)
development
Whole organism > organ > tissue > cell > cellular organelle
Ex) human heart muscle myocyte nucleus
Gross anatomy
microscopic anatomy
(histology)
This lecture is about the microscopic anatomy of the developmental process of teeth.
Methods)
naked eye
light microscopy
electron microscopy

3. Two types of cells give rise to teeth
zygote
Enamel
epithelial cells
Dentin
Pulp
Proliferation
(increase in number)
ectomesenchymal
cells
Cementum
Migration
(change in location)
Two types of embryonic cells undergo proliferation, migration, and differentiation to form teeth.
Differentiation
(specialization)
Ameloblasts forming enamel
Odontoblasts forming dentin
Cementoblasts forming cementum

4. Early embryonic development
<1 wk
~2 wks
Oral Histology and Embryology by Leslie P. Gartner, 1988
bilaminar germ disc
<trilaminar germ disc>
Ectoderm
(tissues covering outside the body / epidermis, hair etc)
Three layers of embryonic cells (ectoderm, mesoderm and endoderm) will be developed to specified tissues in the future.
Mesoderm
(connective tissues / heart, muscle, blood cells, bone, dermis etc)
Endoderm
(tissues lining inside the body / lung, stomach, intestine, liver etc)
~3 wks

5. Tissues originated from ectoderm besides epidermis
epithelium
3. connective tissues of head and neck
(bone, cartilage, muscles, tooth etc)
(Ectomesenchymal origin)
ectodermal origin
embryonic
connective tissue
Ectoderm undergoes invagination to mesodermal side and form two more differentiated structures; neural tube and neural crest. Neural crest gives rise to sensory ganglia and connective tissues of head and neck, including a part of tooth.
Therefore, teeth are originated from two types of cells.
Ectodermal origin  generate enamel
Ectomesenchymal origin  generate dentin, cementum, and pulp
neural crest
2. sensory ganglia
neural tube
1. brain (neuroectodermal origin)

6. Location of tooth formation
6 weeks old embryo
Coronal section
nasal septum
Maxillary process
eye
auricle
tongue
Mandibular process
Tooth formation occurs at the oral epithelium of maxillary and mandibular processes of embryo.
Oral epithelium of maxillary
and mandibular processes
Oral Histology and Embryology by Leslie P. Gartner, 1988

7. Stages of tooth development
1. Bud stage
(epithelial ingrowth into ectomesenchyme)
2. Cap stage
(further epithelial growth)
3. Bell stage
(histo- and morpho-differentiation)
4. Appositional stage (mineralization)
(formation of enamel and dentin of crown)
Tooth develops by complicated continuous processes of proliferation and histomorphological differentiation. For convenience of explanation, the stages of tooth development are separated as presented. However, there is no clear cut between stages.
* The figure only displays changes in the epithelium.
* Note the disintegration of dental lamina in panel d.
5. Root formation
(formation of dentin and cementum of root)
6. Eruption
Oral Histology and Embryology by Leslie P. Gartner, 1988

8. Bud stage Dental lamina - outgrowth of horse shoe-shaped
epithelial band from oral epithelium
- one dental lamina per arch
developing
maxillalry bone
- 10 tooth buds per lamina
palate
tongue Mx
dental lamina
This slide shows the frontal section of the head of ~6 week-old embryo. Thin (within several micrometers) sections were stained by hematoxilin and eosin to reveal the structure under light microscopy. A tooth bud of a mandibular molar is shown.
Bud stage is represented by the epithelial outgrowth.
outgrowth of dental lamina from oral epithelium.
outgrowth of vestibular lamina from oral epithelium.
outgrowth of tooth buds from dental lamina.
tooth bud Mn
developing
mandibular bone

9. Bud stage Mx vestibular lamina Mn vestibule *vestibule
a furrow between gingival and buccal cheek
a junction of freely movable cheek and gingiva attached to alveolar bone. Mn

10. Bud stage ectomesenchymal cells

11. Bud stage condensation of ectomesenchymal cells around tooth bud
Purple dots represent nucleus of cells. Higher density of purple dots represent more dense distribution of cells. Note the higher density of ectomesenchymal cells around the tooth bud.
condensation of
ectomesenchymal cells
around tooth bud

12. Cap stage 3 1. Enamel organ (=dental organ) 2 2. Dental papilla 1
(proliferation of epithelial cells) 2
2. Dental papilla
(condensation of ectomesenchymal cells) 1
3. Dental sac (=dental follicle)
(capsule like condensation of
ectomesenchymal cells)
This is a tooth germ of maxillary tooth. At this stage, epithelial cells proliferate to form enamel organ. Ectomesenchymal cells form two different structures, dental papilla and dental sac.

13. Future tooth products 3 1. Enamel organ 2 2. Dental papilla 1
dentin
pulp 1
3. Dental sac
Each structure will form tooth and periodontal structures in the future.
cementum
periodontal ligament
alveolar bone
connective tissues of gingiva

14. Tooth germ means + + 3 1. Enamel organ 2 2. Dental papilla 1
3. Dental follicle
Tooth germ = enamel organ + dental papilla + dental follicle.

15. Differentiation of enamel organ
Transformation of a cell mass into morphologically and functionally distinct components
Enamel organ
Secreting proteoglycan
(absorbing water  increase extracellular space)
desmosome
Flattened (squamous or cuboidal)
Star shaped
Elongated and polarized (columnar)
Differentiation represent the changes in the properties of the cells. In contrast, proliferation represent the increase in number. At this stage, proliferation and differentiation occur at the same time. Differentiation is obvious in a mass of epithelial cells (enamel organ). Differentiation may start by the secretion of proteoglycan from the epithelial cells to the extracellular space. The absorption of water by the proteoglycan increases the extracellular space. The cells in the epithelial layers are not separated but still maintain contacts between cells via the tight intercellular junction by desmosome. Such changes eventually make the cells look star-shaped. Inner (a side facing dental papilla) enamel epithelium undergoes independent differentiation processes to make them taller and polarized.
The height of the cells : Squamous < cuboidal < columnar
Polarized : the nucleus is located at one side of the cell.
1) Outer enamel epithelium
2) Stellate reticulum
3) Inner enamel epithelium

16. Enamel organ of cap stage3
1) Inner enamel ep
2) Outer enamel ep
3) Cervical loop
4) Stellate reticulum
5) Enamel knot
6) Enamel cord
7) Enamel navel 1 2 4
(inner+outer enamel ep.) 5 6
Inner enamel ep : a layer facing dental papilla. The cells are tall and polarized.
Outer enamel ep : a layer facing mesenchyme. The cells are cuboidal or squamous.
Cervical loop : junction of inner and outer enamel ep.
Stellate reticulum : a mesh like structure composed of star-shaped cells.
Enamel knot : a condensation of stellate reticulum at the deepest part of enamel organ.
Enamel cord : an extension of enamel knot toward outer enamel ep.
Enamel navel : a depression on the outer enamel ep to which enamel cord is directed.
5-7 are transiently observed during cap stage. 7
transient structure
during cap stage

17. Enamel organ of cap stage3
1) Inner enamel ep
2) Outer enamel ep
3) Cervical loop
4) Stellate reticulum
5) Enamel knot
6) Enamel cord
7) Enamel navel 1
generate enamel 2 4
root formation 5
(protect inner enamel ep.) 6
Inner enamel ep is the most important structure since it will generate enamel in the future. Cervical loop will proliferate further to form the shape of crown and eventually guide the formation of root. Stellate reticulum and outer enamel ep might play a role to protect inner enamel ep layer. Enamel knot may be a site where the calcification is initiated and therefore the location of future cusp tip.
location of cusp? 7
transient structure
during cap stage

18. Tooth germ of late cap stage
(tooth bud of
permanent tooth)
* Basement membrane : a structure composed of collagen fibers and attachement proteins that connects connective tissue and epithelium. It is a universal structure between connective tissue and epithelium, not a unique structure of the tooth germ.
Oral Histology and Embryology by Leslie P. Gartner, 1988

19. Bell stage 1. Tooth shape is identifiable.
2. Further differentiation of enamel organ
1) outer enamel ep.
2) inner enamel ep.
3) stellate reticulum
4) stratum intermedium 4 1 2 3
Proliferation and differentiation of epithelial and ectomesenchymal cells continue and form a larger tooth germ with more complicated structures.

20. Bell stage 1. Tooth shape is identifiable.
2. Further differentiation of enamel organ
1) outer enamel ep.
2) inner enamel ep.
3) stellate reticulum
4) stratum intermedium 4 1 2 3
Enamel organ is further differentiated into four distinct layers.

21. stratum intermedium inner enamel ep basement membrane dental papilla
- several layers of cells between the inner enamel epithelium and stellate reticulum
may be derived from the retraction of stellate reticulum.
show exceptionally high activity of alkaline phosphatase, an enzyme essential for mineralization

22. Bell stage
Oral Histology and Embryology by Leslie P. Gartner, 1988

23. Differentiation of inner enamel ep.
differentiated
to ameloblast
without proliferation
Cusp
tip
older
taller
polarized
The cells in inner enamel epithelium at this stage are composed of a spectrum of cells with different degree of proliferation and differentiation.
1. The cells at the cervical loop – mainly proliferating
The ‘proliferation’ of inner enamel epithelium mainly occur at the cervical loop. These cells are younger (recently proliferated and undifferentiated) and the shape is round and short. As the cells proliferate, the cells migrate to the direction of the future root.
2. The cells at the deep part of enamel organ – mainly differentiating
The cells at the deepest part of the enamel organ stop proliferating and migrating. Instead, the cells mainly undergo ‘differentiation’ to prepare forming enamel. The cells become taller and polarized.
younger
shorter
unpolarized
root
Keep proliferating
and migrate
downward

24. cap stage bell stage appositional stage
* Note the relative distance from inner enamel epithelium to outer enamel epithelium become shorter as the tooth germ develops.
* The absolute size of the tooth germ become bigger as the development continues. The presented examples are scaled to the similar size for better contrast of the morphological changes.

25. cap stage bell stage appositional stage
* The absolute size of the tooth germ become bigger as the development continues. The presented examples are scaled to the similar size for better contrast of the morphological changes.

26. Appositional stage Stage of mineralization 1 1. oral ep.
2. outer enamel ep.
3. stellate reticulum
4. inner enamel ep.
5. dental papilla
6. cervical loop 2 3 4
At the appositional stage, the mineralization starts at the deepest part of the enamel organ.
Note that blood vessel does not exist within the enamel organ but outside outer enamel epithelium.
Blood vessels supply oxygen and nutrients required for the mineralization.
The oxygen and nutrients diffuse into outer enamel epithelium and stellate reticulum to reach inner enamel epithelium. Therefore, the distance from the blood vessels to inner enamel epithelium has to be short.
Vascularization close to the tooth germ can be seen in bell stage as well. It increases greatly during appositional stage. 5
blood
vessels 6

27. Epithelial and mesenchymal cells interact throughout tooth formation1
bud
Cap
mineralization
bell
mesenchymal
cells
oral epithelium
dental lamina 1
signal
epithelium
mesenchyme
proliferation
Epithelial and mesenchymal cells interact each other throughout the tooth formation.
1. Mesenchymal cells underlying oral epithelium induce the proliferation of oral epithelium to form dental lamina.

28. Epithelial mesenchyaml interaction1
bud
Cap
mineralization
bell
mesenchymal
cells
oral epithelium
dental lamina
inner enamel epithelium 2
preameloblast 2
preodontoblast
Preodontoblasts
Preameloblasts a 1 a 1 b 2 b 2 c 3 c 3 d 4 d 4
2. Pre-ameloblasts (differentiated inner enamel epithelial cells to form enamel in the future) induce the differentiation of ectomesenchymal cells to preodontoblasts that will synthesize the matrix of dentin. e 5 e 5 f 6 f 6 g 7 g 7 h 8 h 8 i 9 i 9 j 10
mesenchyme
epithelium k 11 l 12 m 13

29. inner enamel epithelium3 1
Predentin (dentin matrix)
bud
Cap
mineralization
bell
odontoblasts
preameloblasts
mesenchymal
cells
oral epithelium
dental lamina
inner enamel epithelium 2
preameloblast
preodontoblast
odontoblast 3
enamel matrix
dentin
Predentin
ameloblasts
3. Dentin matrix (uncalcified predentin) induce the differentiation of preameloblast to ameloblast which synthesize the matrix of enamel.
ameloblast
dentin
enamel matrix
enamel

30. Appositional stage 6 a. predentin b. dentin c. enamel c 1. ameloblasts
2. preameloblasts
3. odontoblasts
4. preodontoblasts
5. dental papilla
6. stratum intermedium c 1 b a
Please remember the order of differentiation.
Preameloblasts  preodontoblasts  odontoblasts  dentin  ameloblasts  enamel 2 3 5 4

31. stratum intermedium ameloblasts enamel dentin predentin odontoblasts

32. Developmental lobes tooth germ of mandibular molar
groove (junction of 2 lobes)
tooth germ of mandibular molar
cusp
Pit (junction of >3 lobes)
Initiation of mineralization
at 5 points
continued mineralization
toward cervical direction
Coalescence of 5 segments
Each tooth has multiple starting points of mineralization. The unit mineralizing segment is called a developmental lobe. Coalescence of lobes results in grooves and pits.
Developmental lobes

33. Biomineralization Dentin = collagen + hydroxyapatite crystals
Concrete = metal framework cement
Dentin = collagen + hydroxyapatite crystals
Enamel = amelogenin hydroxyapatite crystals
resorbable
The formation of tooth structure is initiated by the synthesis of matrix protein (collagen in dentin and amelogenin in enamel). The inorganic crystals are accumulated in the matrix protein to form hard tissues. Note that the matrix of enamel (amelogenin) disappears during the mineralization process to allow dense packing of inorganic crystals. In constrast, collagenous matrix of dentin is not resorbed.
allows further packing of crystals
inorganic component, hardness
enamel >>> dentin

34. Essential components of calcification1 2
Ca2+ + PO43- 3
Alkaline phosphatase (supply phosphate)
Pyrophosphatase (hydrolyze pyrophosphate)
ATP
pyrophosphate
AMP
Ca10(PO4)6(OH)2
Calcium and phosphate form hydroxyapatite crystals. Alkaline phosphatase increases the local concentration of phosphate by the cleavage of organic phosphate at the site of mineralization. Although calcium, phosphate and alkaline phosphatase present abundantly in many tissues, calcification does not occur in every tissue since ‘pyrophosphate’ inhibits calcification even in the presence of calcium and phosphate. Therefore, inhibition of the role of pyrophosphate by pyrophosphatase is also necessary for the mineralization process. The odontoblasts and osteoblasts release matrix vesicles for mineralization. Matrix vesicles contain not only the essential components of calcification but also seeds of inorganic crystals where the growth of crystals can occur easily within organic matrix.
Matrix vesicles
Contain essential components
Contain seed of crystal
Released from odontoblasts to collagen matrix

35. Acid can reverse calcification
Ca2+ + PO43-
acid
acidogenic bacteria
Ca10(PO4)6(OH)2
Any type of acid can reverse the mineralization process easily. The remineralization of the demineralized tooth substances is very limited.
Bone maintains a balance between mineralization by bone-forming cells (osteoblasts) and demineralization by bone-resorbing cells (osteoclasts) in order to maintain physiological concentrations of calcium and phosphate in the body. There is no such mechanism in tooth.
Dental caries
decalcification of inorganic tooth substances by acid produced by bacteria
Only very limited remineralization can occur in tooth.
(cf: bone maintains a balance between mineralization and demineralization )

36. Appositional stage Reduced stellate reticulum Aprismatic dentino-
(alkaline phosphatase )
(predentin)
Aprismatic
dentino-
enamel
junction
(collagen fibers of mantle dentin)
Mantle
This is a sketch of electronmicroscopic structures.
Ameloblasts and odontoblasts are actively synthesizing the matrix to form enamel and dentin. Large part of cytoplasm is filled with cellular organelles, such as endoplasmic reticulum and golgi complex, for synthesizing protein matrix.
Ameloblasts and odontoblasts keep moving away from the dentino-enamel junction as they synthesize matrix. Ameloblasts die away once they synthesize a full thickness of enamel. But odontoblasts remains and continuously synthesize dentin in the pulp.
Stratum intermedium contains high concentration of alkaline phosphatase.
Aprismatic enamel : a layer of first synthesized enamel
Mantle dentin : a layer of first synthesized dentin
Korff’s fiber : collagen fibers of mantle dentin
odontoblasts
Oral Histology and Embryology by Leslie P. Gartner, 1988