this is the brief view of how dentin is formed...the molecular biology and epigenetics of it

1. KUNAL PAREKH

2. Dentinogenesis
Dentin is formed by
odontoblasts that
differentiate from
ectomesenchymal
cells of dental papilla with
influence from the inner
dental epithelium

3. 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.

4. 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.

5. 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.

6. Structure
of mature
secretory
odontoblasts

7. 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.

8. Go l g i c o mp 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.

9. Ba 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.

10. Un i d i r e c t i o n a l
a nt e r ogr a de
v e s i c ul a r t r a ns por t

11. 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.

12. F o r ma t i o n o f c o a t o me r c

13. 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)

14. F O R MA T I O N , D O C K I N G
AND F US I ON OF
T RANS P ORT VESI CL ES

15. 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

16. Od o n t o b l a s t
s e c r e t or y
p a t h wa y
Ctv-intermediate coated transport vesicle.
CGN- cis golgi network
Psg-pre secretory granules
Tgn– trans golgi network

17. 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..

18. S E CRE T I ON OF DE NT I N
MA 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

19. 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.

20. As synthesis of type 1 collagen increases
expression of type 3 decreases.
Dental matrix contain type 1 collagen and variety
of glycoproteins and gylcosaminoglycans.

21. COMPOSITION OF THE DENTIN MATRIX

22. The matrix provides a framework for mineralization.
Collagens comprise 90% of the dentin matrix, and are
principally type I
Ty pe I c ol l a ge 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

23. Ty pe I c ol l a ge 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.

24. Ty pe I I I c ol l a ge 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

25. 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.

26. No n c o l l a g e n o u s
p r ophosphoprotein (DPP; phosphophoryn)
dentin
t e i ns
dentin sialoprotein (DSP)
represent the most abundant dentin-specific acid proteins
in the dental matrix
Odontoblast secret proteoglycans, phosphophoryns and
glycoproteins

27. 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..

28. De n t i n
s i a l ophos phopr ot 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.

29. 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.

30. 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.

31. De n t i n
p h o p h o p r o t e i n , or p h o s p
hophor 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

32. 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.

33. 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.

34. D e n t i n Ma t r i x P r o t e i
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

35. 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.

38. 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 m i c a n , which carry
glycosaminoglycan (GAG) carbohydrate side chains within their
structures, comprise another sizeable portion of the
noncollagenous proteins

39. Fi br one 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.

40. 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).

41. Mi 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..

42. 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.

43. 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.

44. 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.

45. 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.

46. Dentinal tubules
Peritubular dentin Intertubular dentin

47. F O R MA T I O N O F
I NT ERT UBUL AR
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

48. PERI T UBUL AR DENT 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.
.

49. F o r ms o f
de nt 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 A, Primary dentin;
B, Secondary (regular) dentin;
and few dentinal tubules. With aging or
C, Reactive dentin
severe damage, tertiary dentin can
totally obliterate the pulp cavity.

50. 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.

51. AGE & F u n c t i o n a l
Ch 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.

52. 2. R e p a r a t i v e de nt 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.

53.  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.

54. De 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 odontoblast
processes give rise to dead tracts.

55.  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.

56. or , Tr a ns pa r e nt
de nt 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.

57.  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

59. 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 occlusal
dentin, 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.