ENAMEL

PRESENTER: LEKSHMY JAYAN

I MDS,

ORAL AND MAXILLOFACIAL

PATHOLOGY

INTRODUCTION• Ectodermally derived structure produced by ameloblasts

• Hardest substance in the body

• Wear resistant outer layer of the dental crown

• Forms insulating barrier – protects the tooth

[Rodrigo.S.Lacruz et al: Dental enamel

formation and for oral health and disease,

May 2017]

[Michel Goldberg el al : Dentin:

structure, composition and

mineralisation, January 2011]

COMPOSITION (ROBINSON ET AL, 1971)

95%

1% 4%

COMPOSITION OF ENAMEL

INORGANIC ORGANIC WATER

• Inorganic components – various minerals

• Organic components – forms enamel matrix ( non collagenous proteins and enzymes)

• Primary function of organic material- direct growth of enamel crystals.

• Enamel proteins

Non- amelogenins in enamel formation- Ameloblastin, Enamelin, Tuftelin

AMELOGENINS (90%)

NON-AMELOGENINS

(10%)

• Inorganic component – mainly hydroxyapatite crystals, carbonates and trace

elements.

• Enamel hydroxyapatite crystals- largest of all calcified tissues

• Susceptible to dissolution of acid- basis of dental caries.

• Water is present as a part of hydroxyapatite crystal, boundaries of rods.

[ Jayasudha et al: Enamel regeneration- Current progress and challenges,

September 2014]

INORGANIC ORGANIC (ENAMEL PROTEINS)

OXYGEN (43.4%) AMELOGENIN (90%)

CALCIUM (36.6%) NON AMELOGENIN (10%)

PHOSPHORUS (7.7%) o AMELOBLASTIN

SODIUM (0.67%) o ENAMELIN

CARBON (0.64%) o TUFTELIN

MAGNESIUM (0.35%) o AMELOTIN

FLUORIDE, STRONTIUM, LEAD

o ODAM, DSPP

HYDROXYAPATITE CRYSTALS

• Augustin Alexis Damur, 1856

• Naturally occurring mineral form of calcium apatite

• Chemical formula- Ca5 (PO4 )3 (OH)

Ca10 (PO4 )6 (OH)2

• Hydroxyl end member of complex apatite group.

• Hydroxyl group replaced by F,Cl,CO3 –

Fluroapatite or Chlorapatite

• Carbonated calcium deficient hydroxyapatite-

tooth and bone

• Also seen in calcification within pineal gland -

Corpora arenacea (Brain sand)

• CO3 substitution for OH or PO4 – susceptible to

acidic dissolution- progression of caries

• F substitution – resistant to dissolution- caries

prevention and erosion reduction

[Rodrigo.S.Lacruz et al: Dental enamel formation

and implication for

oral health and disease, May 2017]

HYDROXYAPATITE CRYSTALS IN ENAMEL

• Closely packed, long, ribbon like carbonate apatite

crystals

• Width =60-70 nm, Thickness =25-30 nm

• Length span the entire thickness of enamel layer.

• Maturing enamel- hexagonal crystal

• Matured enamel- irregular

Hydroxyapatite crystal

in bone

PHYSICAL CHARACTERISTICS

Protective covering Hardest calcified tissue

Resistant covering Brittle

• Modulus of elasticity- 1338.2+307 MPa

• Hardness – 274.8+18.1 kg/mm

• Specific gravity- 2.8

[ K.J.Chun et al: Comparison of mechanical properties and role between enamel and dentin

in the human teeth, 2014]

• Semipermeable membrane

- demonstrated by radioactive tracers and certain dyes

- (C- labelled urea, Iodine).

- The organic matrix and water in enamel is in a network of micropores-

dynamic connection between enamel and

systemic, pulpal or dentinal tubule fluids

- Micropores or cracks allow the penetration of fluids.

-Permeability decreases and hardness increases with age.

[Jansen et al: Permeability of normal enamel, 1951]

• Colour of enamel – yellowish white to greyish white

determined by translucency

[ K.J.Chun et al: Comparison of mechanical properties and role

between enamel and dentin in the human teeth, 2014]

STRUCTURE OF ENAMEL

• Rods or prisms

• Hunter Schreger bands

• Incremental lines

• Gnarled enamel

• Surface structures

• Enamel tufts and lamellae

• Enamel cuticle

• DEJ

• Enamel cuticle

ENAMEL RODS

• Fundamental organisational units of enamel

• Cylindrical in LS

• Number- 5 million to 12 million

• Length is greater than thickness

• Increased area of enamel in surface than at

DEJ

• Diameter- 4μm

• Clear crystalline appearance

• Arcade outline- hexagonal , round,

oval, fish-scale near DEJ

• Keyhole shape outline near enamel

surface

ULTRASTRUCTURE:

• Key hole pattern or paddle shaped pattern

• Width =5μm Thickness= 9μm

• In LS, sections pass through heads or bodies of one row of rods and tails of an

adjacent row, producing an appearance of rods separated by interrod substance.

• Bizarre pattern- packed tightly together.

Head (1) occlusal and incisal surface

• One rod is formed by 4 ameloblast

Tail (3) cervical region

• EM- apatite crystals, parallel to long axis of

the rods, heads deviate about 65°

• Length- 0.05-1μm Thickness-30nm

Width- 90nm

CROSS STRIATIONS:

• Each enamel rod is built up of segments,

separated by dark lines- striated appearance

• Demarcate rod segments

• More pronounced in hypocalcified enamel

• Formed by diurnal rhythm in enamel

matrix formation

• Length of each segment-4μm

DIRECTION OF RODS:

CLINICAL CORRELATION:

Unsupported Enamel Rods:

-Un supported enamel rods are brittle

and susceptible to fracture-break to

produce leakage at the margins -

lodging of food/bacteria in these

spaces- secondary caries.

• Other patterns that complicate enamel structures:

-Irregular bending in transverse plane of tooth,

cervical- straight

-Intertwine in inner 2/3rd- dissimilar local orientation

- Wavy course in clockwise and anticlockwise direction in

cuspal and incisal edge

- Developmental pits and fissures- converge in outward

course

CLINICAL CORRELATION:

Wavy course of enamel rods:

The wavy course, oblique direction and interlocking of the enamel rods render

prevention from enamel fracture

ROD SHEATH

• Thin peripheral layer

• Darker than rod

• Relatively acid resistant

• Less calcified than rod

• Often incomplete

INTERPRISMATIC SUBSTANCE

• Cement enamel rods together

• More calcified than rod sheath

but less than rod

• Minimum in human teeth

HUNTER-SCHREGER BANDS

• Optical phenomenon seen in LS

• Found in inner 2/3rd of enamel

• More or less regular change in direction of

rods- functional adaptation

• Parazones-dark bands

• Diazones-light bands

• Angle between parazone and diazone-40°

• Enamel crystals aggregate in each zone, deviated in opposite

direction and tilted to 50° with respect to central axis.

• Controversies in the formation of Hunter Schreger bands:

1. Change in the direction between adjacent group of rods

2. Variation in calcification of enamel

3. Composed of alternate zones of different

permeability and different content of

organic material

INCREMENTAL LINES

INCREMENTAL LINES NEONATAL LINE

OF RETZIUS

INCREMENTAL LINES OF RETZIUS

• Brownish bands in GS of enamel calcified teeth

and in forming enamel

• Illustrate incremental pattern of enamel

• LS- surround tip of dentin

• Cervical part- run obliquely from DEJ to

surface, deviate occlusally

• Transverse section- concentric circle

• Represents 6-11 days of rhythmic deposition of

enamel

• Other proposed causes:

1. Periodic bending of enamel rods

2. Variations in basic organic structure

3. Physiologic calcification rhythm

MEAN DAILY FORMATION OF ENAMEL = 4μm

CLINICAL CORRELATION :

Accentuated incremental lines can also be pathological,

caused by metabolic and systemic disturbances such as

exanthematous fever that affects enamel formation.

NEONATAL LINE OR RING• Boundary marked between enamel formed

before and after birth

• Accentuated striae of Retzius

• ETIOLOGY

- Sudden change in environment and nutrition

- Antenatal enamel is better calcified than

postnatal enamel

• Used to identify enamel formed before and

after birth

• Seen in all deciduous teeth and in

permanent first molars

• Frequently seen in first molars of girls than

boys

• Location also varies in pre and post term

birth

GNARLED ENAMEL• Wavy pattern in the enamel at the cuspal

region

• Optical appearance of enamel cut in

oblique plane

• Bundle of rods intertwine more regularly

• Makes enamel more stronger

• Not hypomineralised!

SURFACE STRUCTURES

• Prismless enamel

• Perikymata

• Enamel pits and caps

• Cracks or enamel lamellae

• Enamel tufts

• Rod ends

PRISMLESS ENAMEL• Relatively structureless layer of enamel,

approximately 30μm thick

• Seen in 70% of permanent and all deciduous

teeth

• Most commonly in cervical areas of enamel

surface

• Surface, prismless, hydroxyapatite parallel to

each other and perpendicular to Striae of

Retzius

• Hypermineralised

PERIKYMATA (IMBRICATION LINES)

• Transverse, wave like grooves, external

manifestation of Striae of Retzius

• Lie parallel to each other and CEJ

• 30 per mm in CEJ

• 10 per mm near occlusal or incisal edge of a surface

• COURSE- regular, irregular in cervical region

ENAMEL ROD ENDS

ENAMEL PITS AND CAPS

• Enamel pits- 1-1.5μm in diameter, depressed ends of ameloblast

• Caps- small elevation of about 10-15μm, enamel deposition on non-

mineralizable debris

• Enamel brochs- large enamel elevations

ENAMEL LAMELLAE• Thin, leaf like structures that extend from the enamel

surface toward the DEJ

• May extend or penetrate dentin

• Consist more of organic and less of inorganic

• Confused with cracks

• Develop in areas of tension- when rods cross, donot

calcify

• Disturbance severe- crack develop – filled by surrounding

cells or organic substance from oral cavity

• TYPES OF ENAMEL LAMELLAE

• Cells from enamel organ fill a crack in

enamel

- in depth degeneration

- close to surface, remain vital for

sometime – HORNIFIED CUTICLE

From connective tissue- cementum

formation

CLINICAL CORRELATION-

SITE OF WEAKNESS!! – pathways for

cariogenic bacteria

ENAMEL TUFTS• Resembles tufts of grass in GS

• Arise at DEJ and reach into the enamel to

about 1/5th to 1/3rd of its thickness

• Narrow, ribbon-like structure, inner end

arises at the dentin

• Tufts in different planes are projected into

one plane- TUFT OF GRASS

• Extent in direction of long axis of the crown

• Hypocalcified enamel rods and

interprismatic substance

• SEM- tubular structure with cross striation

• TEM- plate like structure in centre of tufts

originating from superficial layer of dentin,

crossing DEJ and entering tufts

ENAMEL CUTICLE

• PRIMARY ENAMEL CUTICLE- Nasmyth’s membrane

• SECONDARY ENAMEL CUTICLE- Afibrillar cementum

• PELLICLE- Precipitate of salivary protein

ENAMEL CUTICLE (NASMYTH’S MEMBRANE/ PRIMARY ENAMEL CUTICLE)

• Covers entire crown of newly erupted tooth

• Removed by mastication

• Basal lamina secreted by ameloblasts when enamel formation

is complete

• Protects enamel surface from resorption by adjacent vascular

tissue prior to eruption of teeth

SECONDARY ENAMEL CUTICLE

• Cover cervical area of the enamel

• Thickness=upto 10μm

• Continuous with cementum

• Probably of mesodermal origin or may be elaborated by

attachment epithelium

• Secreted after enamel organ is retracted from cervical

region during tooth development

PELLICLE

• Reform within hours after mechanical cleaning

• May be colonised by microorganisms to form a bacterial plaque

• Plaque may be calcified forming calculus

CLINICAL CORRELATION-

If not removed, the pellicle can get colonized by microbes to form

plaque which subsequently lead to caries.

DENTINOENAMEL JUNCTION

• Surface of dentin at DEJ –pitted, depression fit

rounded projection of enamel, holds the enamel

firmly on the dentin.

• Scalloped, convexity towards dentin

• Crystals of enamel and dentin mix with each other

• Series of ridges- more pronounced in occlusal area-

greater masticatory stress

• Hypermineralised zone about 30μm thick at DEJ-

prominent before mineralisation is complete

ENAMEL SPINDLES• Odontoblastic process that cross the DEJ into enamel,

thickened at their end before mineralisation

• Hypomineralised

• Direction corresponds to direction of ameloblast(90°

• to dentin)

• Enamel rods and spindles are divergent

• GS- dark in transmitted light

• Width=5nm Length=70nm Diameter=2μm

• Seen in cusp tip

AGE CHANGES

• Attrition

• Generalised loss of enamel rods

• Flattening of perikymata

• Decreased permeability

AMELOGENESIS

AMELOGENESIS• Enamel first forms, 30% mineralised

• Organic matrix breaks down and removed – crystals grow wider and

thicker- 96% mineralisation

• Ameloblast secrete matrix protein

• Ameloblast has unique lifecycle- phenotypic changes

• Enamel formation – differentiation of IEE, OEE – cuspal tips – all cells

are differentiated into ameloblast

• Dentin and enamel formation cuts off blood supply to enamel organ

• Reversal of nutritional source

LIFECYCLE OF AMELOBLAST

MORPHOGENIC STAGE

• Ameloblast- short, columnar, large oval

nuclei ,almost fill the cell body

• Terminal bars appear during

differentiation

• Migration of mitochondria to basal

region of the cell

• IEE separated by dental papilla basal

lamina

• Adjacent pulp layer- cell free, narrow,

light zone

ORGANISING STAGE• IEE interacts with adjacent connective tissue

which differentiates into ODONTOBLASTS

• Reversal of polarity

• Cell free zone between IEE and dental papilla

disappear

• Preameloblasts secrete protein similar to enamel

matrix- phagocytosed by odontoblast-

EPITHELIAL MESENCHYMAL INTERACTION

• Terminal phase- DENTIN FORMATION,

reversal of nutritional source

FORMATIVE STAGE• After dentin formation

• Presence of dentin is necessary for formation of

enamel

(Reciprocal induction)

• Formation – enamel matrix retain same length and

arrangement

• Intiation – secretion of enamel matrix- change in

organisation and number of cytoplasmic organelles

and inclusion

• Earliest change- development of blunt cell

processes ameloblast surfaces

MATURATIVE STAGE• Maturation/ full mineralisation after most of the

thickness of enamel matrix formed in occlusal/

incisal area, cervical area – progressing

• Ameloblast-slightly reduced in length and closely

attached to enamel matrix

microvilli at distal extension, cytoplasmic

vacuoles containing enamel matrix like material

(absorptive function)

• Cells of stratum intermedium- assume spindle

shape

• Smooth and Ruffle ended ameloblasts

PROTECTIVE STAGE• Enamel fully developed and calcified

• Ameloblast- indistinguishable from OEE and

stratum intermedium

• OEE + AMELOBLAST + STRATUM

INTERMEDIUM = REE

• REE protect mature enamel, separates it from

connective tissue till eruption

• Retraction of enamel organ from cervical edge

CLINICAL CORRELATION-

Contact occurs –afibrillar/ coronal cementum

formation on enamel or resorption

DESMOLYIC STAGE

• REE – separates oral epithelium and CT

• Elaborate desmolytic enzymes- destroy CT fibres

• Premature degeneration of REE- prevent eruption of tooth

1. FORMATION ENAMEL MATRIX• Secretory activity after dentin deposition

• Lose projection penetrating basal lamina

(separation between predentin and

ameloblast)

• Islands of enamel matrix deposited along

predentin

• Continuous layer of enamel formed along

dentin

ENAMEL MATRIX PROTEINS:

AMELOGENIN(1983)- Major component

Extracellular degradation by MMP – tyrosine and leucine rich amelogenins

Regulate cell growth

Genes coding present in both X and Y chromosome

Most of amelogenins secreted is removed during maturation

Maintain space between enamel crystals

Remains between and around the crystals

CLINICAL CORRELATION-

AMELOGENESIS IMPERFECTA-Mutations in human

Amelogenin gene located on the X chromosome (AIH1)

-hypoplastic or hypomineralized.

KALLIKREIN-4 - Secreted in late enamel formation by ameloblast

Remove Amelogenin scaffold prior to mineralisation.

CLINICAL CORRELATION-

1. Over expression- Hypocalcified enamel

AMELOBLASTIN(1996)- Nucleation and growth of crystals

Cell-matrix attachment

Maintenance of ameloblast in differentiated state

CLINICAL CORRELATION-

1. Lack of expression- Termination of amelogenesis

Failure to produce any enamel.

ENAMELIN(1997)- Original enamel protein

CLINICAL CORRELATION-

1. Lack of expression- No true enamel formed

Thin, highly irregular mineralised crust covered the dentin

AMELOTIN – New protein – secreted by mature ameloblast

Late stage of enamel formation

CLINICAL CORRELATION-

1. Over expression- Extremely soft enamel

hypomineralisation of inner enamel and structural defects in outer enamel.

TUFTELIN- Localised to DEJ

Involved in cell signalling

Odontogenic Ameloblast associated gene(ODAM) (2006)-

Secreted in late secretory, transition stage, maturation stage

CLINICAL CORRELATION-

1. Lack of expression- No enamel defect but altered junctional epithelial attachment

Predispose tooth to periodontal infection

DENTIN SIALOPHOSPHOPROTEIN- Localised to DEJ

Transient in ameloblast and in odontoblast till dentin formation is complete

Proteolytic cleavage into Dentin Sialoprotein and dentin Phosphoprotein

Regarded as transition zone between dentin and enamel

CLINICAL CORRELATION-

1. Over expression DSP- Increase enamel hardness

DPP- Soften and weakened bulk enamel

NBCe1- Electrogenic sodium bicarbonate cotransporter

Also seen in renal proximal tubule, pancreas, eye, heart, brain, developing tooth.

Ameloblast- maintain pH buffering during mineralisation

CLINICAL CORRELATION-

1. Lack of expression- Enamel too soft to even measure

Dentin is significantly softened

DEVELOPMENT OF TOMES’ PROCESS• Projection of ameloblast into enamel matrix

• Partly delineated by incomplete septa

• Junctional complex encircle ameloblast at proximal and

distal ends – form webs

controls substances that pass between ameloblast

and enamel

• Distal terminal bars – separate Tomes’ process from cell

proper

• Secretion from areas close to junctional complex and

adjacent ameloblast – INTERROD ENAMEL

• Distal portion of Tomes’ process lengthens and narrower

AMELOBLAST COVERING MATURING ENAMEL

• Shorter than ameloblast over incompletely formed

enamel

• TRANSITION STAGE- changes occurring in

ameloblast after secretory stage and prior to onset

of maturation process

Ameloblast decrease in

size

Enamel secretion

stops

Amelogenin removal

starts

Phagocytosis of ameloblasts

(50%)

Autophagocytosis of organelles

• During Maturative stage,

ameloblast cycle between

RUFFLED and SMOOTH

border – MODULATION-

every 5 to 7 hours/ day

• Period of maturation more

than secretion

Ruffle-ended Ameloblasts Smooth-ended Ameloblasts

Leaky proximal and tight distal junctions

Tight proximal and leaky distal junctions involved in exchange of molecules

Numerous lysosomes exhibit considerable endocytosis

Little endocytic activity

Presence of organelles which promote pumping of calcium ions into maturing enamel

No calcium pumping activity

CLINICAL CORRELATION-1. Enamel hypoplasia2. Chronological Enamel Hypoplasia

Ameloblast secretes proteases of different types :

MMP

• Serine proteases

• Membrane bound proteins present in ameloblast – CD63, annexins A2,

lysosomal associated glycoprotein 1 – removal of organic matrix

• 90% of protein lost during maturation, remaining protein envelopes around

individual crystals

MINERALISATION AND MATURATION OF ENAMEL MATRIX

1. IMMEDIATE PARTIAL MINERALISATION OF MATRIX

2. MATURATION

IMMEDIATE PARTIAL MINERALISATION OF MATRIX

• Occurs in matrix segments and interprismatic substance as they are laid

down

• No matrix vesicles, no unmineralised matrix

• No apatite crystal formation

• Initiation of nucleation by hydroxyapatite crystals of dentin

• Initial mineral- OCTACALCIUM PHOSPHATE

MATURATION• Gradual completion of mineralisation

• Starts from height of crown progresses cervically, from dentinal end of the rods

• Integration of 2 processes- 1. Each rod matures from depth to surface

2. Sequencing of maturing rods from cusp

towards cervical line.

• Begins before matrix has reached full thickness

• Matrix deposited on inner surface, mineralisation on outer surface of recently

deposited matrix

• Incisal and occlusal region mature ahead of cervical region

ULTRASTRUCTURE:

• Growth of crystals

• Increase in size from about 1.5-25μm during maturation phase

• Tuftelin- nucleation of enamel crystals

• Other proteins- regulate mineralisation, binding to specific surface of crystal , further

deposition

• RATE OF FORMATION =4μm/day

1mm thickness= 40days

• Loss of organic matrix is caused by withdrawal of protein and water

WHY IS AMELOGENESIS UNIQUE?

FEATURES AMELOGENESIS DEVELOPMENT OF OTHER MINERALISED STRUCTURE

IN TOOTH

SECRETORY CELLS Epithelial Ectomesenchymal

MINERALISATION By non collagenous protein Collagen has main role

MATRIX • Lacks collagen• 90% absorbed by ameloblast

• Collagen is the main protein

• No absorption

MINERALISATION OF MATRIX DURING

FORMATION

Partial No

ORGANIC PHASE Absent Present (Osteoid, Predentin, Cementoid)

REGENERATION No- ameloblast undergo apoptosis

Regenerate throughout life

CLINICAL CONSIDERATION

• ENAMEL HYPOPLASIA

• ENAMEL HYPOCALCIFICATION

• FLUOROSIS

ENAMEL REGENERATION• Enamel cannot regenerate or remodel on its own

• Various methods are employed to attempt regeneration of enamel

• Synthetic enamel fabrication , tissue engineering etc

• Can done by

a. Hydrothermal method- Controlled release of Ca from Ca-EDTA

b. Hydrothermal transformation- Octacalcium phosphate rod to Hydroxyapatite

nanorods and using hydrogen peroxide containing pastes

c. PAMAM-COOH solution- organic template on demineralised enamel produce

hydroxyapatite crystals

Skin epithelial

cells

Oral keratinocytes

Human embryonic stem cell derived

epithelial cells

Bone marrow cells

Epithelial cell rests of

Malassez

Alternative cell sources for enamel formation are-

[ Jayasudha et al: Enamel regeneration- Current progress and challenges,

September 2014]

ENAMEL BIOMIMETICS

• Biomimetic methods

• Enamel acts like a single crystal

• Replication of Enamel Translucency- should regenerate the highly organised structure

of enamel

• Fundamental difficulty in clinical application- low solubility of calcium phosphate

- difficult to remineralise deep lesion

• Crystal precipitate randomly- will not rebuild enamel structure- develop stabilising

agents

CASEIN

CHITOSAN GEL

EDTA

Example of some enamel biomimetic materials

[Rodrigo.S.Lacruz et al: Dental enamel formationand implication for oral health and disease, May 2017]

REFERENCES

Rodrigo.S.Lacruz et al: Dental enamel formation and implication for oral health and

disease, May 2017

Jayasudha et al: Enamel regeneration- Current progress and challenges, September 2014

Michel Goldberg el al : Dentin: structure, composition and mineralisation, January 2011

Yue Sa et al : Compositional, structural and mechanical comparisons of normal enamel and

hypomaturation enamel, August 2014

Rick J. Rauth et al: Dental Enamel: Genes Define Biomechanics, December 2009

J.P. Simmer et al: Molecular mechanisms of dental enamel formation, 1995

Jansen et al: Permeability of normal enamel, 1951

Orban’s Oral Histology and Embryology(13th Edition): G.S.Kumar

Tencate’s Oral Histology, Development, Structure and Function(8th

Editon):Anonio Nanci

Textbook of Oral Anatomy, Histology, Physiology and Tooth Morphology

(1st Edition) : Dr K. Rajkumar, Dr. R. Ramya