Amelogenesis

Dr. Gábor Varga

Department of Oral Biology

February, 2016

Amelogenesis - introduction

• Amelogenesis as a part of tooth formation

• Secretory phase of amelogenesis

• Maturation phase of amelogenesis

• Proteins involved in amelogenesis

Molar

longitudinal

section

the enamel

covers the dentin

Pulp Horn

LAMINA BUD STAGE CAP STAGE BELL STAGE ERUPTION

Tooth development

Gene activation during tooth development

Epithelium

Mesenchyme

Tooth development – details 1

Tooth development – details 2

Section of tooth – enamel and dentin formation

Formal and structural changes of ameloblasts

during enamel formation

1 2 3 4 5 6 7

1. morphogenetic, 2. inductive, 3. early secretory, 4 secretory,

5. maturation - ruffle-ended, 6. maturation – smooth-ended, 7. protective

Amelogenesis

1st - Secretory phase

Secretion of proteins

Foundation of the mineral structure

2nd – Maturation phase

Reabsorption of proteins and water removal

Secretion of mineral ions

The initiation of enamel formation on the surface of

the already formed, unstructured mantle-dentin

zománc=enamel .

The arrangement of ameloblasts during enamel formation

Outer enamel epithelium

Stellate reticulum

Stratum intermedium

Ameloblasts

Enamel matrix

Fully differentiated secretory ameloblasts

Secretory ameloblasts and surrounding cells

Ultrastucture

of secretory

ameloblasts

Tomes process sorrounded by freshly produced enamel

SG – secretory granule, PZ – prismatic (rod) enamel, IPZ –

interprismatic (interrod) enamel

Enamel

structure

Proximal

Distal

PE

IPE

IPE

PE

PE

N

GA

SG

TP

Sh Sh

TP

N: Nucleus

GA: Golgo apparatus

SG: Secretory granule

TP: Tomes process

Sh: Sheath region

PE: Prismatic enamel

IPE: Interprismatic enamel

Secretory ameloblasts –

formation of prismatic enamel

(PE) and interprismatic enamel

(IPE)

Three dimensional arrangement of crystal rods

(prismatic enamel) in the vincinity of Tomes processes

Parallel running crystallites (Kr) in the early phase

of enamel development

Amelogenesis

• 1st - Secretory phase

• 2nd – Maturation phase

2.a. reabsorption of proteins

and water removal

2.b. secretion of mineral ions

Papillary layer (PL) cells between the capillaries

and the maturation ameloblasts (MA).

Basement

membrane

Enamel

Dentin

Transitions Multiple

Za & Zo

M

M

E

G

Maturation ameloblast phenotypes: ruffle-ended and smooth-ended

maturation ameloblasts cycle back and forth during the maturation

phase

Ruffle-ended and smooth-ended maturation ameloblasts cycle back and forth

during the maturation phase. Cycling of the two phenotypes involves extensive

remodeling of the distal cytoplasm and junctional complexes at both ends of

the cells. The Golgi complexes (G) and the lysosomal (L) apparatus are well

developed in both cell configurations. Zonula adherens (Za) and zonula

occludens (Zo) shift from distal position in the ruffle-ended ameloblasts to a

proximal position in the smooth-ended ameloblasts. Mitochondria (M) are

located primarily in the distal cytoplasm. Endosomes (E) containing enamel

matrix are present both in the ruffle-ended and smooth-ended ameloblasts. The

ruffle ended surface primarily supports electrolyte exchange while the smooth

ended form is for cell recovery and protein absorption.

The two types of ameloblasts during the absorptive phase

Mineral secretion Protein and water absorption

Ultrastructure

of ruffle-ended

maturation

ameloblasts

CFTR

Na+

Basolateral

membrane

Apical

membrane

Tight junction

Na+

H+

Na+

2HCO3−

Na+

K+

2Cl−

H+

Na+

NHE1

NBCe1

(?) NKCC1

CaCC

NHE3 (?)

HCO3−

Cl−

CO2 + H2O (?)PMCA Ca

2+

AE2

~ 3Na+

2K+

Na-K-

ATPase K

+ (?) Maxi-K

K+

TASK2 (?)

CA 2HCO3−

Cl− PAT-1

Ameloblast transporters

CO2 CO2

H+

H+-ATPase ~

Ca2+/ K+

Na+

NCKX4

Ca2+

Na+

NCX

10 Ca2++ 6 HPO42-+ 2 H2O ↔ Ca10(PO4)6(OH)2+ 8 H+

Cl-(HCO3-)

(?)

Na+

Basolateral

membrane

Apical

membrane

Tight junction

Na+

H+

Na+

2HCO3−

Na+

K+

2Cl−

H+

Na+

HCO3−

Cl−

CO2 + H2O PMCA Ca

2+

~ 3Na+

2K+

K+

K+

CA 2HCO3−

Cl−

Ameloblast calcium transport elements

CO2 CO2

H+

~

Ca2+/ K+

Na+

NCKX4

Ca2+

Na+

NCX

10 Ca2++ 6 HPO42-+ 2 H2O ↔ Ca10(PO4)6(OH)2+ 8 H+

Cl-(HCO3-)

(?)

CFTR

Na+

Basolateral

membrane

Apical

membrane

Tight junction

Na+

H+

Na+

2HCO3−

Na+

K+

2Cl−

H+

Na+

NHE1

NBCe1

(?) NKCC1

CaCC

NHE3 (?)

HCO3−

Cl−

CO2 + H2O Ca

2+

AE2

~ 3Na+

2K+

Na-K-

ATPase K

+ (?) Maxi-K

K+

TASK2 (?)

CA 2HCO3−

Cl− PAT-1

Ameloblast bicarbonate transport elements

CO2 CO2

H+

H+-ATPase ~

Ca2+/ K+

Na+

Ca2+

Na+

10 Ca2++ 6 HPO42-+ 2 H2O ↔ Ca10(PO4)6(OH)2+ 8 H+

Cl-(HCO3-)

(?)

Ameloblast bicarbonate transport elements

Car 2

Car 9

2 HCO3- + 2 H+ 2 H2O

+ 2 CO2 Na+

Nbce

H+ HCO3-

H2O + CO2

Na+

Nhe1

Ae2 HCO3

-

Cl-

Cl-

Cl-

HCO3-

H+

PD Cftr

CO2

H2O

Apatite formation

Bicarbonate is generated by carbonic anhydrase 2

(Car2) and is exchanged by the pendrin (PD) anion

exchanger in the apical membrane for Cl-. Cl- is

imported basolaterally by Ae2 and conducted into

the enamel by CFTR.

Car 2 = carbonic anhydrase 2 (cytosolic),

Car 9 = carbonic anhydrase 9 (membrane-bound),

Cftr = cystic fibrosis transmembrane conductance regulator

Ae2 = anion exchanger 2,

Nbce1 = sodium bicarbonate exchanger 1,

Nhe1 = sodium hydrogen exchanger 1,

P D= pendrin: HCO3-/Cl- exchanger,

█ = tight junction

Basolateral

Apical

Hypothetic model for pH regulation by ruffle

ended ameloblasts to neutralize liberated H+

Proposed pathway of enamel protein

reabsorption and digestion by ruffle-ended

ameloblasts

B

1 2 3 4 5 6 7

A

Tj

M

M

End

G

End

pH cycling in rodent incisior ameloblasts

pH cycling in rodent incisior ameloblasts

Damkier at al. Bone, 60, 2014, 227 - 234

Hypothesis on the dynamics of phosphate equilibrium in solution

and enamel crystal (A) and the effect of ameloblasts on phosphate

dynamics in the RA phase (B) and SA phase (C).

Damkier at al. Bone, 60, 2014, 227 - 234

Rod enamel (prismatic enamel, PZ) in cross section

electron microscopy picture

Cross sectional scanning electron microscopy

picture following acidic treatment

Longitudinal sectional electron scanning microscopy

of the enamel – rods are well visible

Amelogenesis - enamel proteins

• Amelogenin

• Enamelin

• Ameloblastin

• Amelotin (Ben Ganss, Toronto)

• Tuftelin

• Osterix (Ben Ganss, Toronto)

• Proteinases (enamelysin - MMP-20 kallikrein 4 – KLK4)

• Phosphatases

Proteins with know function are in bold

Amelogenin

1

2

3 4

5

Amelogenin

secretion

Assembly Hydrophylic

anionic terminals

exposed

Nanospheres act

as spacers

between

crystallites

Platelike crystallites

of hydroxyapatite

Proteinase- 1

(enamelysin)

removes hydrophylic

tails

Nanospheres

hydrophobic

Proteinase-2

degrades the

nanospheres

resorption

Crystals

grows in

thickness

Concept of the role of amelogenins in the

mineralization of enamel The hydrophobic amelogenesis form globular aggregates (nanospheres) on

secretion into the extracellular space. The nanospheres form lattices that

regulate the spacing and the orientation of the C-axis of the newly forming

enamel crystallites

Disorder scores of amino

acid sequences of

proteins participating in

biomineralization

Disorder frequency of amino acid chains of

proteins participating in various biological

functions

Distribution of amelogenin and

ameloblastin in enamel matrix

Defect of amelogenesis in ameloblastin-

null mice

p75

Msx2 Amelogenin

Amelogenin

p21, p27 Regulation of cell cycle

Trks

?

Receptor?

Enamel crystal

Ameloblastin in the enamel matrix

Ameloblast

Role of ameloblastin in the regulation of

ameloblast function

Amelogenesis imperfecta

Amelogenesis imperfecta

The human amelogenin gene

Amelogenin mutation leading to hypoplasia

- loss of three amino acid and substitution of

another one

56 66 48 42 45 435 160 56 66 48 42 45 435 160

1 2 3 4 5 6 7 1 2 3 4 5 6 7

Hypoplastic

Amelogenesis imperfecta

(X-linked)

Hypomineralization

Amelogenesis imperfecta

(X-linked)

Genomic

sequence

mRNA

Predicted

protein

Phenotype Thin enamel Poorly mineralized enamel

9 bp deletion 5 kb deletion

1 2 3 4 5 6 7 1 2 4 5 6 7 3

The bar segments represent the introns and the boxes (1 through 7)

correspond to the exons. The nucleotide numbers are indicated below the

exons. (Adapted from Simmer et al.)

Structure of the X-chromosomal copy of the

human amelogenin gene

Two mutations of the amelogenin gene that

cause amelogenesis imperfecta

Amelogenin (AMELX) mutations causing

X-linked amelogenesis imperfecta

How are changes in the AMELX gene related

amelogenesis imperfecta? • One copy of the amelogenin gene is located on each of the sex chromosomes (the X and

Y chromosomes). The AMELX gene, which is located on the X chromosome, makes

almost all of the body's amelogenin. The copy of the amelogenin gene on the Y

chromosome, AMELY, makes very little amelogenin and is not needed for enamel

formation.

• At least 15 mutations in the AMELX gene have been identified in people with X-

linked forms of amelogenesis imperfecta. (X-linked disorders are caused by mutations

in genes on the X chromosome.) Some AMELX mutations lead to the production of an

abnormal version of the amelogenin protein that can interfere with the formation and

organization of enamel crystals. Other AMELX mutations prevent one copy of the gene

from producing any amelogenin protein at all. Enamel cannot form properly without an

adequate amount of amelogenin

• Males have a single copy of the X chromosome in each cell. Males who inherit a

defective copy of the AMELX gene have very little amelogenin and develop almost no

enamel to cover and protect their teeth. Females have two copies of the X chromosome

in each cell. Females who inherit one altered copy of the AMELX gene are less severely

affected because they have a normal copy of the gene on the other X chromosome to

produce amelogenin. Their tooth enamel may have structural defects such as a

distinctive pattern of vertical grooves. No symptoms other than abnormal enamel

development have been reported in people with AMELX mutations.

Enamelin mutations causing autosomal

dominant amelogenesis imperfecta

Enamelysin (MMP20) and kallikrein 4

(KLK4) mutations causing autosomal

recessive amelogenesis imperfecta

Proteins of enamel involved in Amelogenesis Imperfecta

Amelogenin: (product of AMELX and AMELY genes located on the X and Y chromosomes) is the most

abundant protein in developing enamel [26, 27]. While its exact role in enamel formation is not fully

understood, it is thought to be crucial for regulating the size and shape of the mineralizing enamel

crystallites. Multiple human mutations in the AMELX gene are associated with different AI types. There are

no known AMELY mutations. A transgenic mouse lacking expression of this gene has only a very thin

covering of enamel that lacks a prismatic structure [28].

Ameloblastin: (product of AMBS gene located on chromosome 4) is another enamel associated protein that

appears to be the second most abundant enamel matrix protein [29]. The function of this protein is mónot

completely known but it may regulated ameloblast differentiation and formation. It is considered a likely

candidate for being associated with some AI types.

Enamelin: (product of ENAM gene located on chromosome 4) is secreted by amelobasts in relatively low

amounts. It is speculated that this protein could interact with amelogenin or other

enamel matrix proteins and be important in determining growth of the length of enamel crystallites.

Three different mutations ENAM gene mutations are associated with different AI types.

Enamelysin: (MMP20 gene located on chromosome 11) is a proteinase that cleaves amelogenin and is

thought to be the major proteinase involved in processing the enamel matrix proteins [32, 33]. The

enamelysin knockout mouse has a reduced enamel thickness and the enamel lacks a prismatic

structure.

Kalikrein 4: (KLK4 gene located on chromosome 19) is a proteinase that is secreted predominantly

during the maturation stage of enamel development [34]. This aggressive proteinase could be

responsible for processing any proteins not cleaved by enamelysin.

Period of

amelogenesis in

the permanent

teeth of human

dentition.

Bars: from

beginning to

completion.

Amelogenesis - summary

• Amelogenesis as a part of tooth formation

• Secretory phase of amelogenesis

• Maturation phase of amelogenesis

• Proteins involved in amelogenesis