ABOUT DIFFERENT THEORIES OF MINERALIZATION OF HARD TISSUES OF ORAL CAVITY

1. 1

2. Resource Faculty: Dr. Ashish Shrestha
Dr. Vinay Marla
Presented by:
Roshan Yadav(640)
Roshna Sunar(642)
THEORIES OF
MINERALIZATON
2
Tuesday, April 18, 2017

3. CONTENTS
3
 HARD TISSUE
 ORGANIC MATRIX
 MINERALS
 MINERALIZATION
 THEORIES OF MINERALIZATION

4. HARD TISSUE
4
 Mineralized and have firm intercellular
substances.
 Includes bone, cementum, dentin and enamel
 Except enamel , they are all
-Specialised connective tissue
-Collagen (esp. type 1) plays a role in
determining their structure.
 HARD TISSUE FORMATION: Cells-production of
organic matrix-capable of accepting mineral +
activity of alkaline phosphatase + a good blood
supply prerequisites

5. FORMATIVE CELLS
5
 Ability to synthesise and secret organic matrices of hard
tissues
 Fibroblast
 Odontoblast
 Ameloblast
 Cementoblast
 Osteoblast
 They all possess :
 Abundant mitochondria
 Golgi apparatus
 Rough endoplasmic reticulum
 Transport vesicles and secretory vesicles.

6. ORGANIC MATRIX
6
 Collagenous proteins:
 Type I collagen : It acts as scaffold that accumulate the
minerals in holes and pores of fibrils.
 Non - collagenous proteins:
 Proteoglycans
 Phospholipids
 Phosphoprotein
Non collagenous protiens are involved in mineralization of
enamel whereas in other hard tissues collagen play an
important role.

7. ORGANIC MATRIX OF
ENAMEL7
consist of a distinctive family of enamel proteins:
 90% amelogenins
 Proline, Histidine, Glutamine
- helps to maintain space between crystals.
 10% non- amelogenins
 Tuftelin, enamelin, amelin
-helps in nucleation and growth of crystals.
Nevertheless, all hard tissues regardless of their composition
are capable of accepting minerals in form of hydroxyapatite
crystals.

8. ENAMEL DENTIN FIBRILAR
CEMENTUM
BONE
ORGANIC
MATRIX
AMELOGENIN
NON-
AMEOGENIN
COLLAGEN
NON-
COLLAGENO
US
COLLAGEN
NON-
COLLAGENO
US
COLLAGEN
NON-
COLLAGENO
US
STRUCTURE GLOBULAR RANDOM
FIBRILS
FIBRILS(BUN
DLES &
SHEETS)
RANDOM
FIBRILS(=)
STATUS DEGRADED
WITH
AMELOGENIN
S
REMAIN IN
MATRIX
REMAIN IN
MATRIX
REMAIN IN
MATRIX
MINERAL HA>90% HA>67% HA>45-50% 50-60%
LOCATION AMELOGENIN
S
COLLAGEN COLLAGEN COLLAGEN
NUCLEATION CONTROVER
SIAL
MATRIX
VESICLES
MATRIX
VESICLES
MATRIX
VESICLES
FORMATIVE AMELOBLAST ODONTOBLA CEMENTOBLA OSTEOBLAST

9. MINERALS
9
 Inorganic component of mineralized tissue
 Consist of mainly: calcium hydroxyapatite i.e a biological
apatite. Ca10(PO4)6(OH)2
 Unit cell - least no. of Ca, phosphate and hydroxyl ions
able to establish a stable ionic relationship.
 Shape-hexagonal
 Unit cells stacked together - lattice of crystal
 Various size – number of repetition of this arrangement
 HA crystal in mesenchymal hard tissue:100*200*50*50A
 HA crystal in enamel: length – 1400A ; width – 800A

10. BIOLOGICAL APATITE
10
Biological apatite is built on a definite ionic lattice pattern that
permits considerable variation in its composition through
substitution, exchange and adsorption of ions.
 In general,
 Shape of apatite crystals – needle like or platelike.
 Shape of apatite crystals in enamel – long, thin ribbons.
 Each apatite crystal has 3 surfaces:
 Crystal interior
 Crystal surface
 Hydration shell
All of 3 surfaces are available for exchange of ions.

11. CONTD..
11
 Exchange of ions can take place between:
 Mg & Na can substitute Ca position
 F and Cl can substitute hydroxyl position
 Carbonate in both hydroxyl & phosphate position.
 F substitution decrease the solubility of crystallites
whereas carbonate increase.
 Mg inhibits crystal growth.
 Adsorption of ions - electrostatic attraction or bound in
hydration layer.
 The apatite crystal is able to retain its structural
configuration while accomodating these substitution.

12. 12
 Deposition of mineral salts in an around organic
matrix to make it a calcified structure.
 Although , tissue fluid is supersaturated with Ca & P
ion , spontaneous precipitation of calcium phosphate
does not take place.
 BECAUSE:
 Inhibitory macromolecules-inhibit crystal formation.
 Unstable-initial cluster of ions needed to form a lattice
structure
 Furthermore, the formation of clusters of ions
requires the expenditure of energy and an energy
barrier must be overcome for crystallization.
MINERALIZATION

13. CONTD..
13
 When deposition is initiated, the crux is then to:
 Control spontaneous precipitation from tissue
fluids
 Limit it to well defined sites
 This function is done by formative cells by:
 Creating microenvironment facilitating mineral ion
handling
 Secreting proteins that stabilizes Ca & P ions in
body fluids
 Controlling Ca & P ions deposition on to ECM.

14. NUCLEATION
14
 HOMOGENOUS NUCLEATION :
Any local increase in concentration of
inorganic ions permits a sufficient no. of ionic
clusters & crystallite to form.
 HETEROGENOUS NUCLEATION:
The presence of nucleating substance allows
crystal formation to occur, in absence of a locally
increased ionic concentration.

15. MECHANISM OF
MINERALIZATION15
 For initiation:
1. Matrix vesicle
2. Heterogeneous nucleation/Collagen
mineralization
 For continuation:
1. Collagen growth
2. Secondary nucleation

16. 1.MATRIX VESICLES
16
 Small membrane bound structure lying free in the matrix
 Size= 25-250nm in diameter
 Rounded outgrowth of cell membrane , buds off from
osteoblasts,chondrocytes and odontoblasts
 Exist only in relation to initial mineralisation
 Form an independent unit within the first form organic matrix.
 Rich in phospholipids(esp. phophotidyl serine)-high affinity for
Ca ions.
 A site for Ca and Pi accumulation by deposition of initial
mineral complex (i.e. nucleation) occurs & hydroxyapatite is
produced.
 Annexins in vesicles form a Ca channel thus incorporating
ions.

17. CONTD..
17
Alk. phosphatase , pyrophosphatase, Ca ATPase, metalloproteinases,
proteoglycans & anionic phospholipids bind Ca & P ions
Calcium inorganic phosphate phospholipids complex
Initiate mineralization.
Crystallites grows rapidly & rupture from vesicle
Fuses with adjacent clusters - form mineralized matrix

18. Fig: 1st:Matrix vesicle containing apatite crystals.
2nd:Crystals have ruptured from vesicles &
are joining with others to form mineralized
tissues.
18

19. 2.HETEROGENOUS
NUCLEATION19
 Apatite crystals deposited in the surface,holes
& pores of collagen.
 But collagen itself have no role in it’s initiation.
 The non- collagenous protein fulfil this function
, although their role in mineralization process
is complex and not fully understood.

20. COLLAGEN
MINERALIZATION20
 Collagen acts as a nucleating substance (which
has the effect of lowering the energy barrier)that
allows crystal formation to occur even in absence
of locally increase ionic concentration.
 In the gap zones at the ends of collagen
molecules , the mineral first appears.
Initially, proteoglycans that bind with Ca are filled in
gap zones
PGs are now removed enzymatically by
proteoglycanase enzyme leaving behind the Ca

21. CONTD..
21
Phosphoprotein bind with the collagen
Immobilizes the phosphate and initiate 1st mineral
deposit
Alkaline phosphatase causes dephosphorylation of
phosphoproteins
Localized increase in Pi encourage the precipitation of
additional Ca phosphate complexes in the gap zones.
Rapidly convert into 1st hydroxy apatite crystal

22. CONTD..
22
Eventually hydroxyapatite crystals spread between
collagen fibrils to fully mineralize tissues.
Exception:
 Neither of these mechanisms is involved in mineralisation of
enamel.
 Since matrix vesicle and collagen is absent in enamel.
 Initiation of enamel mineralisation is thought to be achieved
by crystal growth from the already mineralised dentin , by
matrix proteins secreted by the amelobasts .
 Shape & size of crystals determined by enamel proteins of
matix
 Mineral nucleation can also occur in relation to
noncollagenous matrix proteins

23. Fig. Collagen mineralization
23

24. 1.CRYSTAL GROWTH
24
 Initial mineralisation is rapid.
 Later slower growth exceed their initial size by 10-
20 times.
 Important factors influence crystal growth &
composition is it’s immediate environment of
growing crystal
 Non- collagenous proteins bind selectively to
different surfaces of crystal ,preventing further
growth and thereby influence final size of crystal.
 Pyrophosphate accumulation on crystal surface
also blocks further growth.

25. 25
NAME COMPOSITIO
N
LOCATION POSSIBLE
FUNCTION
NONCOLLAGENOUS PROTEIN
OSTEOPONTIN
OSTEONECTIN
PHOSPHOPHORY
N
BONE
SIALOPROTEIN
II
GLA PROTEIN
(OSTEOCALCIN)
PHOSPHOPROTEI
N
PHOSPHOPROTEI
N
PHOSPHOPROTEI
N
PHOSPHORYLAT
ED
GLYCOPROTEIN
PROTEIN & y-
BONE
,DENTIN
,CEMENTUM
DENTIN
EXCLUSIVE
DENTIN
BONE,DENTI
N
,CEMENTUM
BONE,DENTI
N, CELLULAR
CEMENTUM
Inhibit crystal growth.
Inhibit crystal growth.
Low concentration
induces hydroxyapatite
formation; high
concentration inhibit
crystal growth.
Nucleator for
mineralization.
Regulator for crystal
growth.

26. B
A
C
Fig.1
A
B
C
Fig.2
A
B
C
Fig.3
No inhibition
(block shaped
crystal)
Inhibition of the B
face alone (plate
shaped crystal)
Inhibition of growth of A and B faces(needle shaped crystal)
Eg: noncollagenous proteins are able to bind
selectively to various surfaces of crystal which
prevent further growth & thereby determining
shape of crystal
26

27. 2.SECONDARY NUCLEATION
27
 Additional crystallites may form by secondary
nucleation from mineral-phase particles arising
from collision and fracture of crystals
previously form by heterogeneous nucleation.

28. TRANSPORT OF MINERAL IONS
TO MINERALIZATION FRONT28
1. Intercellular transport
 Tissue fluid is supersaturated in these ions so
fluid simply needs to percolate between cells to
reach organic matrix
 More likely to occur between osteoblasts and
odontoblasts
 In case of enamel,secretory stage ameloblasts
restrict the passage of calcium, so majority of
calcium entry occur through transcellular route.
 Sequestration of Ca to Golgi appratus,
Mitichondria &ER is a safety device to control
calcium concentration of cytosol

29. CONTD..
29
2. Transcellular transport:
Occurs only if cytosolic free Ca ion
concentration not exceed 10^(-6)M
2 mechanism :
i)Enters cell through specific Ca channels
& sequestered by Ca binding protein to the
site of release
ii) Continuous & constant flow of Ca ions
occurs across cell

30. LOCATION OF MINERAL
30
 Minerals are not simply packed between the
collagen fibrils but also within it.
 In bone: 70-80% of mineral is located within
the collagen fibril.
 Location of such mineral is the result of
heterogeneous nucleation, followed by
secondary nucleation within the gaps of
collagen fibrils.

31. 31

Fig: Localization of mineral within collagen
fibrils

32. ALKALINE PHOSPHATASE
32
 Key enzyme in the process of mineralization .
 In hard connective tissue, found either associated with
matrix vesicles or occurring freely within matrix.
 Capacity to cleave phosphate ions from organic
substrates at alkaline pH,increasing its conc. &
leading to deposition of apatite.
 Ion handling when associated with cell membrane
 Extracellular activity:
 Helps in continuation of crystal growth by inhibiting
pyrophosphate.
 Cleavs pyrophosphate to inorganic phosphate
 No activity is observed in ameloblasts.

33. THEORIES OF
MINERALISATION33
1. Robinson’s phosphate(Alkaline Phosphatase)
theory
2. Nucleation theory (Seeding theory)
3. Matrix vesicle theory

34. 1. Alkaline Phosphatase
Theory34
 Alkaline phosphotase resides in matrix vesicle
 It increases the conc. of ions to such a level
that leads to its precipitation.
 Enzyme hydrolyses the organic phosphate
containing substrate and increases the local
inorganic phosphate conc.
 Enzyme hydrolyses PPi (inhibitor of HA) &
provide Pi for the formation of HA crystals.

35. 2. Nucleating Theory
35
 A nucleus formed in relation to collagen effective in
aggregating Ca & phosphate ions and grow to form
HA by addition of ions from supersaturated fluids
Possible seeding substances:
a. Ground substances : Glycosaminoglycans and
proteoglycans.
b. Collagen :
 Initial mineral deposits at discrete sites in or on the
collagen fibrils
 Allow ingress of ions, formation of ion cluster & its
aggregation to form nuclei for crystal growth
Phosphoproteins also indudce apatite formation.

36. 3. Matrix vesicle Theory
36
 Matrix vesicle accumulte Ca ions & its
membrane furnish binding sites for nucleation of
HA crystals.
Vesicles formed:
1.Type 1:
 Small membrane bound , budding off from cell
membrane as an independent unit within the 1st
formed organic matrix.
 It helps in initiation of mineralization
2. Type 2: Cell degradation
3. Type 3: Extrusion of intracytoplasmic vesicles

37. ENAMEL MINERALIZATION
37
 Cells secrete enamel proteins which immediately
participitate in mineralizing enamel(30%)
 Bulk removal of enamel proteins and water makes
space for growth of crystal(95%)
 No matrix vesicles are associated in
mineralization
 No unmineralized matrix like that of predentin or
osteoid is seen
 Therefore apatite crystals are not preformed when
they are released by the secretory granules.
 Nucleation is initiated by apatite crystal of dentin
on which enamel is laid .

38. LET’S
SUMMARISE
38

39. 39

40. REFRENCES
40
 ORBAN’S ORAL HISTOLOGY AND
EMBRYOLOGY 13TH EDITION
 TENCATE’S ORAL HISTOLOGY 5TH AND 8TH
EDITION
 WIKIPEDIA

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ANY QUESTIONS