KUNAL PAREKH

Dentinogenesis

Dentin is formed by

odontoblasts that

differentiate from

ectomesenchymal

cells of dental papilla with

influence from the inner

dental epithelium

Differentiation of odontoblasts(withdrawal from the cell cycle, cytological

polarization, and secretion of predentin/dentin)

Odontoblast precursor migrate from neural crest

and become part of ectomesenchymal cell .

During cap stage pre-odontoblast is

concentrated adjacent to inner enamel

epithelium and exit the cell cycle and

differentiate befor pre-ameloblasts of IEE stop

dividing.

REQUIREMENT OF INITIAL ODONTOBLASTIC

DIFFERENTIATION

1.Fibronectin rich substratum.---

fibronectin receptors (165-kDa protein) –

adherence appears to stabilize

cytoskeletal elements , promotes preodontoblast

polarization , and trigger other cytoplasmic process

associated with differenciation .

it also serve as reservoir for growth factors.

2. Aperiodic fibrils for regulating differentiation.

2. Aperiodic fibrils for regulating differentiation.

3. Transforming growth factor β 1 ( TGF - β 1 )

Bind to fibronectin ,

Inhibits cell proliferation ,promoter of odontoblast differenciation,

Matrix synthesis.

4.Calcium –

calcium ions signals for mediating restructuring of cytoskeleton during

estalishment of of odontoblast shape ans polarity towards the IEE.

5. Enamel matrix protein –

are endocytosed in coated vesicle at odontoblast cell surface.

Structure

of mature

secretory

odontoblasts

The intercellular spaces contain collagen fibres ,aperiodic

microfibrils, proteoglycans and fibronectin.

These intercellular fibres (von korff fibres) pass into

predentin between adjacent odontoblast at interruptions of

fascia occludens and fascia adherens junctions.

The RER is the major cytoplasmic organelle within active

odontoblast ..the parallel cistern occupy the supranuclear

cytoplasm.

G o l g i c o m p l e x …

Display morphological ( forming face )

functional polarity ( mature face)

Presecretory granules consisting of type 1

procollagen , glycoproteins, and glycoaminoglycans

develop from cisterns of mature face.

B a s i c s c i e n c e c o r r e l a t i o n ..

the complex cytoplasmic machinery operating in the

golgi complex for targeting secretory proteins to their

appropriate final destination.

Proteins are synthesized in RER and futher

transported to golgi complex where it is

posttranstionally modified ,sorted and packeged for

further transport to either secretory granules, primary

lysosome ,or cell membrane.

U n i d i r e c t i o n a l a n t e r o g r a d e

v e s i c u l a r t r a n s p o r t

Glycosyltransferase and glycosidases contained in the golgi

cisternae sequentially decorate the peptide backbone of the

protein by addition of the carbohydrate side chain..

1. Addtion of oligosaccharides by nitrogen linkage to asparagine..

2. Oxygen linkage at serine and threonine residues

( 2 steps process…..1. addition of N-acetyl-galactosamine

2. addition of galactose and sialic

acid

The budding process require recruitment and attachment of

specific coat proteins on the parent cisternal membrane to form

a mechanochemical “patch” capable of deforming the

membrane into a seprate vesicle.

Coatomer recruitment requires ATP, Ca2+ ,guanosine

triphosphate (GTP) ANS SEVERAL cystolic proteins.

F o r ma t i o n o f c o a t o me r c o a t e d me mb r a n e

Sorting products to appropriate destinations

requires specific signals to control the

docking of transport vesicles with the

correct target compartment..

This is accomplise by transmembrane

protein that act as surface markers.

They are soluble N-ethylmaleimide-

sensitive fusion attachment protein

receptors (SNAREs)

F O R M A T I O N , D O C K I N G A N D F U S I O N O F T R A N S P O R T V E S I C L E S

Attachment of v-snare to t-snare is monitered

and stabilized by Rab-GTP molecule present in

donor vesicle membrane.

With the help of fusion protiens

NSF-P ---- N-ethylmaleimide sensitive fusion

protein

SNAPs---- soluble NSF attachment protiens

O d o n t o b l a s t

s e c r e t o r y

p a t h w a y

Ctv-intermediate coated transport vesicle.

CGN- cis golgi network

Psg-pre secretory granules

Tgn– trans golgi network

Secretory granules formation involves of condensation of

secretory product from larger condensing vacuoles

(presecretory granules ) .

Budding of membrane from the condensing vacuole continues

untill a smaller and denser secretory granules is formed..

intact microtubular system forms a radiating network

extending from the centrosome outward the cell periphery to

translocate granules to specific region of cell surface.

Further transport toward plasma membrane is dependent on

myosin

While in constitutive pathway products are

immediately exported..

S E C R E T I O N O F D E N T I N M A T R I X

Preameloblast and preodontoblast express matrix

metalloproteinase 2 , an enzyme that degrade

collagen and fibronectin ,coincident with the

removal of the basal lamina .

After break up from basal lamima this newly

differntiated odontoblasts adhere to adjacent

odontoblast by stable gap junction and macula

adherens type .permitting ions and small

metabolite to cross from odontoblast to

odontoblast.

odontoblast grow in length and develop large

amount of rough endoplasmic reticulum (RER).

A prominent golgi complex develops in the

supranuclear cytoplasm facin the IEE.

in addition to increase expression of messenger

RNA for collagen type 1,

also express for osteocalcin, dentin phosphophoryn

, and high level of alkaline phosphates.

As synthesis of type 1 collagen increases

expression of type 3 decreases.

Dental matrix contain type 1 collagen and variety

of glycoproteins and gylcosaminoglycans.

COMPOSITION OF THE DENTIN MATRIX

The matrix provides a framework for mineralization.

Collagens comprise 90% of the dentin matrix, and are

principally type I

T y p e I c o l l a g e n is

composed of two identical α1(I) chains and one

α2(I) chain, and a glycine in every third amino acid

position in an individual chain is needed for the

formation of a triple helix structure.

Proα2(I) mRNA has been shown to be expressed

by mature human odontoblasts

T y p e I c o l l a g e n is synthesised as a larger procollagen, which

contains extensions at both the N- and C-terminal

ends, called the aminoterminal and carboxyterminal

propeptides,

which prevent premature collagen aggregation into

fibrils.

After procollagen secretion from cells, extracellular

modification takes place, and propeptides are

removed by specific proteinases and mature

collagen molecules aggregate into a fibrous matrix

which then serves as a support for mineral

deposition.

T y p e I I I c o l l a g e n ,

a homopolymer of three α1(III) chains,

In addition, there is strong evidence that calcified tissues

are also able to express type III collagen,

Type III procollagen has been observed to be transiently

located in human predentin during matrix formation, but not

in mineralized dentin

some expression of type V has been observed in the

predentin of mature human teeth but not in dentin .

Instead, type VI was detected both in predentin and dentin

of intact teeth and it has also been found in the teeth of

dentinogenesis imperfecta patients.

N o n c o l l a g e n o u s

p r o t e i n sdentin phosphoprotein (DPP; phosphophoryn)

dentin sialoprotein (DSP)represent the most abundant dentin-specific acid proteins

in the dental matrix

Odontoblast secret proteoglycans, phosphophoryns and

glycoproteins

Predentin protogylcan

functions is to regulate the size and orientation of

collagen fibril and also control the time and site of

mineralisation either by sequestering calcium or by

shielding potential mineral nucleation sites in the

matrix.

Decorin –

a chondroitin-dermatan sulfate proteogylcan

with binding affinity for type 1 collagen..

D e n t i n s i a l o p h o s p h o p r o t e i n I

s the only protein produced uniquely by odontoblasts, the cells that

produce tooth dentin.

DSPP protein is processed by proteases into several functional fragments;

DSP, DPP and others.

These domains play unique biological functions during

dentinogenesis. Preliminary data showed that

1). BMP2 induced DSPP expression.

2). MMP-9 specially catalyzes DSP into the NH2-terminal and

COOH-terminal fragments. The NH2- and COOH-terminal

fragments of DSP show a clear difference in tooth distributions.

3). The NH2-terminal and COOH-terminal domains bind to their

receptors, integrin 26 and CD105, on cellular membrane.

the transcriptional regulation, posttranslational modification and

signal transduction of DSPP are important for controlling the

initiation, rate and extent of dentin biomineralization.

Processing of dentin sialophosphoprotein (DSPP) (57). DSPP is the precursor of

dentin phosphoprotein (DPP), a phosphoprotein unique to dentin. DSPP is

processed by proteases (BMP-1, MMP-20, MMP-2) into N-terminal dentin

sialoprotein (DSP), intermediate dentin glycoprotein (DGP) and C-terminal DPP.

DPP is adsorbed on hydroxyapatite crystals and is deposited in dentin matrix.

D e n t i n p h o p h o p r o t e i n , or p h o s ph o p h o r y n ,

is important in the regulation of mineralisation of dentin

Phosphophoryn is the most acidic protein ever discovered

and has an isoelectric point of 1.

This extreme acidity is achieved by its amino acid sequence.

Many portions of its chain are repeating -D-S-S- (aspartic

acid-serine-serine) sequences. In protein chemistry, net

acidity equates to negative charge. Being highly

negative, dentin phosphoprotein is able to attract large

amounts ofcalcium

Characteristic sequences in dentin phosphoprotein. Most of the DPP molecule is

composed of repetitive sequences, DSS repeats (above). A unit of this sequence

consists of 1 aspartic acid and 2 phosphoserines. The DSS repeat sequences are rich in

negative charges and provide binding sites for many calcium ions (below) and

for hydroxyapatite crystals.

SIBLING-family genes. The SIBLING family is a family of acidic glycoproteins

present in bone and dentin. The genes of this family are present as a gene

cluster at the 4q22 site of human chromosome 4. SPARCL1: SPARC-like protein

1, DSPP: dentin sialophosphoprotein, DMP1: dentin matrix protein 1, BSP; bone

sialoprotein, MEPE: matrix extracellular phosphoglycoprotein, OPN:

osteopontin. Two genes of enamel matrix proteins are also present near this

gene cluster; AMBN: ameloblastin, ENAM: enamelin.

D e n t i n M a t r i x P r o t e i n 1 (D M P 1)

DMP1 appears to belong to the family of dentin

matrix proteins rich in serine and aspartic acid

and has many potential phosphorylation

sites, especially for messenger-independent

kinases of the casein kinase II group.

DMP1 could possibly regulate the expression of

osteocalcin and alkaline phosphatase.

DMP1 is a calcium binding protein as demonstrated by

calcium binding assay

that DMP1 can nucleate the formation of hydroxyapatite in

vitro in a multi-step process that begins by DMP1 binding

calcium ions and initiating mineral deposition.

P o r c i n e p r e d e n t i n e ma t r i x —Contains active neutral metalloproteinases (56 and 61 kDa

gelatinases ans 25 kDa proteoglycanase) capble of degrading

proteoglycans at mineralisation site.

Activity is clcium dependent..

Proteoglycans, such a s

d e c o r i n , b i g c l y c a n , f i b r o mo

d u l i n a n d l u mi c a n , which carry

glycosaminoglycan (GAG) carbohydrate side chains within their

structures, comprise another sizeable portion of the

noncollagenous proteins

F i b r o n e c t i n

is re-distributed during odontoblast polarization and

interacts with cell-surface molecules.

A nonintegrin 165-kDa fibronectin-binding

protein, transiently expressed by odontoblasts, is

involved in microfilament reorganization.

Growth factors (TGFβ1,2,3/B M P 2,4, a n d 6),

stimulates cytological but not functional differentiation of odontoblasts:

The two events can thus be separated.

Immobilized TGFβ1 (combined with heparin) induced odontoblast

differentiation.

Only immobilized TGFβ1 and 3 or a combination of FGF1 and TGFβ1

stimulated the differentiation of functional odontoblasts over extended

areas and allowed for maintenance of gradients of differentiation.

Presentation of active molecules in vitro appeared to be of major

importance; the BM should fulfill this role in vivo by immobilizing and

spatially presenting TGF(3s).

M i n e r a l i z a t i o n

1.) Matrix vesicles in mantle dentin..

bud from tip of the odontoblastic cytoplasmic process.

It initiate mineralization by concentrating calcium and phosphate ions.

As ion increase hydroxyapatite crystallizes along the inner surface of matix

vessicle surface..

Calcium bonding phospholipids serves as templete for hydroxyapatite

precipitation..

Continue crystal growth ruptures the vesicle and release the hydroxyapatite

crystals into extracellular matrix..

2.) C o l l a g e n p h o s p h o p h o r y n

complexes…

Extracellular Kinases .phosphorylate dentin phosphophoryn which further

linked to collagen fibril,,

This act as nucleators of hydroxyapatite crystal in late mantle dentin and

circumpulpal dentin.

Zone of initial mineralization—• dot like mineral nuclei are aligned parallel to and superimposed on

collagen fibrils.

• Mineral nuclei positioned over hole region of collagen fibrils, suggesting

that the DPPs are bound to collagen at those sites.

• Dental sialoproteins and proteogylcans act as nucleating agents for

perifibrillar hydroxyapatite crystals.

Nucleation of hydroxyapatite by acidic matrix proteins immobilized on insoluble collagen

matrix. Some acidic matrix proteins, e.g. dentin phosphoprotein, have an affinity to

collagen. The surface of the insoluble collagen matrix provides loci to reduce interfacial

energy for nucleation. Calcium ions are bound to the acidic groups of the acidic

proteins, and inorganic phosphates are attracted by the calcium ions. The ionic complex

thus formed may constitute a crystal nucleus.

Control of crystal shape by the acidic matrix proteins. Some acidic matrix proteins, e.g.

dentin phosphoprotein, have affinity to a specific face (e.g. the (100) face) of

hydroxyapatite crystals. These proteins are potent inhibitors of crystal growth.

Their specific adsorption results in the inhibition of growth perpendicular to the adsorbed

face. For example, if the (100) face is covered by proteins, growth in the direction of the a-

axis will be inhibited. As a result the crystal will preferentially grow in the direction of the c-

axis.

S T R U C T U R E S ..

1.DENTINAL TUBULES..

extend from mantle dentine to predentine.,across the

thickness of circumpulpal dentin..

dentinal tubules contain serum proteins including fibrinogen

,albumin , and immunoglobins.

this proteins are carried into the tubules in dentinal fuild

,where they may be come trapped in the lamina limiitans or

bound to the mineral phase of dentin.

Dentinal tubules

Peritubular dentin Intertubular dentin

F O R M A T I O N O F I N T E R T U B U L A R

1. Greatest bulk of mineralised circumpulal dentin.

2. it is formed by the mineralisation of predentin.

3. Matrix is rich in type 1 collagen fibrils

P E R I T U B U L A R D E N T I N .

These inner organic lining is described as a thin organic lining high in GAG and relatively free in collagen fibrils .

Bone sialoprotein and osteonectin have been localized in peritubular dentin.

Because of its small crystallites and non collagenous nature of its organic material , it is more susceptible to demineralization and degradation during the caries process..

F o r ms o f

d e n t i n•Primary dentin, with straight

tubules, is laid down before completion

of the apical foramen.

•Regular secondary dentin is

characterized by a slower rate of

deposition and an abrupt change in the

direction of the dentinal tubules.

•Tertiary or irregular secondary (also

called irritation, reparative or reactive)

dentin is laid down in response to an

irritation or damage to the overlying

dentin and/or enamel.

•This dentin has irregularly arranged

and few dentinal tubules. With aging or

severe damage, tertiary dentin can

totally obliterate the pulp cavity.

A, Primary dentin;

B, Secondary (regular) dentin;

C, Reactive dentin

Interglobulardentin in globular

layer -ground section

•Between the mantle and

circumpulpallayers is a layer of dentin

in which the calcified globules do not

fuse evenly.

•This is called the globular layer.

•In a ground section of dentin, the

less-calcified areas of dentin appear

as irregularly shaped crescents called

interglobulardentin.

A G E & F u n c t i o n a l C h a n g e s

The main changes in dentin associated with aging are

1. Increase in peritubular dentin.

2. Increase in Dentinal sclerosis.

3. Increase in the number of dead tracts.

4. The dentinal permeability decreases with advancing

age.

5. The color of dentin becoming darker with age.

6. Hardness of dentin increases with age, primarily due

to increases in mineral content.

2. R e p a r a t i v e d e n t i n

If the provoking stimulus caused the destruction of the original odontoblasts, the new, less tubular, more irregular dentin formed by newly differentiated odontoblast like cells is called Reparative dentin.

In such dentin the tubules are usually not continuous with those of secondary dentin.

Unlike primary or secondary dentin, which forms along the entire pulp-dentin border, tertiary dentin is produced only by cells directly affected by the stimulus.

The quality (or architecture) and the quantity or degree of tertiary dentin produced are related to the cellular response initiated, which depends on the intensity or duration of the stimulus.

Tertiary dentin may have regular tubules continuous with those of secondary dentin, tubules sparse in number and irregularly arranged or no tubules at all.

The cells forming territory dentin either line its surface or are included in the dentin. In the latter case, this dentin is as referred to as Osteodentin.

Various factors associated with cavity preparation and restoration can influence the tertiary dentinogenic response:

The method of cavity preparation, the dimensions of the cavity, the residual dentin thickness (RDT) of the cavity, etching of the cavity, and the nature of the dental materials used and the method of their application for the restoration.

D e a d t r a c t s In dried ground sections of normal dentin the odontoblast

processes disintegrate, and the empty tubules are filled with air.

These are called Dead tracts.

They appear black in transmitted light and white in reflected light.

Loss of odontoblast processes may also occur in teeth containing dental pulp as a result of caries, attrition, abrasion, cavity preparation, or erosion.

Their degeneration is often observed in the area of narrow pulpal horns because of crowding of odontoblasts.

Dentin areas characterized by degenerated odontoblastprocesses give rise to dead tracts.

These areas demonstrate decreased sensitivity and

appear to a greater extent in older teeth.

Dead tracts are probably the initial step in the

formation of sclerotic dentin.

S c l e r o t i c D e n t i n o r , T r a n s p a r e n t d e n t i n :

Sclerotic dentin results from aging or mild irritation such as slowly advancing caries and causes a change in the composition of the primary dentin.

The peritubular dentin becomes wider, gradually filling the tubules with calcified material, progressing pulpally from the DEJ.

These areas are harder, dense, less sensitive and more protective of the pulp against subsequent irritations.

Sclerosis resulting from aging is physiologic dentin sclerosis.

Sclerosis resulting from a mild irritation is Reactive dentin sclerosis.

It appears transparent or light in transmitted light and dark in reflected light.

The amount of sclerotic dentin increased with age and is most common in the apical third of the root and in the crown midway between the DEJ and the surface of the pulp.

Because sclerosis reduces the permeability of dentin, it may help to prolong pulp vitality.

Eburnated dentin: Refers to the outward (exposed) portion of reactive sclerotic dentin, where slow caries has destroyed formerly overlying tooth structure, leaving a hard, darkened, cleanable surface.

1. Hardness of sclerotic dentin is approximately 30% higher than normal dentin with equal depth, showing this to be a more mineralized tissue;

2. The thickness of the hybrid layer formed on sclerotic dentin is less than normal dentin, thus, showing this tissue to be more resistant to demineralization caused by acid etching;

3. Bond strength to sclerotic dentin is not as high as normal dentin;

4. At the time of acid etching the sclerotic occlusaldentin, doubling the time of application of 35% phosphoric acid contributes to obtaining a bond strength similar to normal dentin; and

5. For normal occlusal dentin, no difference exists in bond strength when 35% phosphoric acid etchant is applied following the manufacturer’s suggested time (15 seconds), or when the time is extended to 30 seconds.