This ppt s describes about Minerals Mineralization Theories of mineralization Booster theory nucleation theory matrix vesicle theory Clinical consideration

1. MINERALIZATION

2. Content
• Minerals
• Mineralization
• Theories of mineralization
Booster theory
nucleation theory
matrix vesicle theory
• Clinical consideration
• References

3. MINERALS
• Chemical elements required by living
organisms other than C, H, O, N
• Naturally occurring in foods and must be
taken in the diet
• Comprise of 4% of the body weight
• Classified according to the amount needed
by the body

4. • Macrominerals- required in large
proportions by the body (≥100mg)
• Calcium
• Phosphorus
• Magnesium

5. • Microminerals (Trace elements)- required
in relatively small amount by the body
• Iron
• zinc
• cobalt
• manganese

6. calcium
• Total body Ca content in
adults is about 1-1.5 kg, of
which 99% exists as the
hydroxyapatite
[Ca10(PO4)6 (OH)2]
crystal in the mineral
phase of bone.
• The remaining 1% of total
body Ca is in soft tissue
and the extracellular fluid
(ECF) space including
blood.
Murray J. Favus, David A. Bushinsky, and Jacob Lemann Jr. Regulation of Calcium, Magnesium, and
Phosphate Metabolism. American Society for Bone and Mineral Research. 2006;76-83.

7. Dietary requirements
• Adult men and women-
800mg/day
• Children(1-18yrs) – 800-
1200mg/day
• Infants(<1yr) – 300-
500mg/day
• Sources
• Milk and milk products
• Beans
• Leafy vegetables
• Fish
• Cabbage
• Egg yolk

8. • Functions:
 Development of bones and teeth
 Muscle contraction
 Blood coagulation
 Nerve transmission
 Membrane intigrity and permeability
 Activation of enzymes – lipase, ATPase,
succinate dehydrogenase
 Release of harmones

9. Calcium absorption
• Promoting factors
 Vitamin D
 Parathyroid harmone
 Acidity
 Lactose
 Amino acid lysine and
arginine
• Inhibiting factors
 Phytates and oxalates
 Phosphate free fatty
acids
 Alkaline condition
 Dietary fibres

10. • Importance of Ca:P ratio
• For calcification of bones
• Ca:P ratio in
Children – 50%
Adults - 40%

11. • Inadequate intake, impaired absorption
and increased loss include:
–Incomplete calcification of teeth
–Tooth and bone malformations
–Increased susceptibility to dental caries
–Increased tooth mobility and premature
tooth loss
–Decreased bone mineral density
(cementum and dentin)

12. PHOSPHOROUS
• Adult body contains
about 1kg phosphate.
• 85% - bones
• 15% - muscle, blood and
chemical compounds
• 0.1% - ECF
Murray J. Favus, David A. Bushinsky, and Jacob Lemann Jr. Regulation of Calcium, Magnesium, and
Phosphate Metabolism. American Society for Bone and Mineral Research. 2006;76-83.

13. • SOURCES
 Milk
 Cereals
 Leafy vegetables
 Meat
 eggs

14. • Functions
 essential for development of bones and teeth
 formation of phospholipids, phosphoproteins
and nucleic acids
 activate several enzymes by phosphorylation
Maintaince PH in blood

15. MINERALIZATION
• DEFINITION:
• It is the process of deposition of minerals
in the organic matrix, which is capable of
accepting the minerals.
• Important step in formation of hard tissue
of the body.

16. What are biologic apetite?

17. • Calcium and phosphates have a strong affinity
for each other and therefore form stable
compounds which have structural and
physiological importance in many living
organisms. Collectively they are referred to as
biological apatites.
• Name derived from the Greek "apatite" to
deceive.

18. • It is a very hard salt and almost insoluble
in water.
• Crystals of apatite form the bulk of the
mineralised part of hard tissues like bones
and teeth.

19. • The crystals are not pure apatite; they include
carbonate,
citrate,
sodium, and
magnesium,
in amounts of about 1% each.
• There are small amounts of fluoride and traces
of heavy metals.

20. • Most common form
hydroxyapatite
(Ca10(PO4)6OH2), but
there are other apatites in
which the calcium and
phosphate parts are in
different ratios.
• Ratio of Ca : PO4 - 1.67,
the highest of all the
apatites and the most
insoluble

21. • CA:P ratios of some apatites
• Monocalcium Phosphate - 0.5
• Dicalcium phosphate - 1.0
• Octocalcium phosphate - 1.3
• Tricalcium phosphate - 1.5
• Hydroxyapatite -1.7
• ACP Alkaline calcium phosphate - 2.0

22. Production of apetite crystals
•

23. • Firstly,
Ca + and PO4 - must accumulate in such
concentration as to exceed the solubility of an
apatite salt and to precipitate.
• Secondly,
the ions must precipitate in a specific pattern
which will allow other ions to spontaneously
arrange themselves in the proper orientation
• third stage, crystal growth .

24. • The [Ca] x [HPO4] product in tissue fluid
is around 1.76 mmol2.
• This means that hydroxyapatite cannot
precipitate without a local catalyst or
template, but once nucleation has
occurred crystals can grow rapidly in body
fluids.

25. • Mineralization can occur at following
circumstnces:
• Homogenous nucleation
• Heterogenous mineralization

26. • Homogenous nucleation
local increase in concentration of minerals
Formation of sufficient ionic crystallites
required for mineralization

27. • Heterogenous mineralization
o In the presence of a nucleating substance
that can act as a template for crystal
formation therefore decreasing the energy
required for mineralization.
o Nucleating substance can initiate
mineralization even in the absence of
increase in ionic concentration

28. • Two types of mineralization
• By organic component
• By cells

29. By organic components
• The matrix in which mineralisation takes place contains
collagen,
proteoglycans,
citrates,
lipids and
plasma constituents.
• The cells concerned all contain large quantities of the
enzyme alkaline phosphatase.

30. • There have been two principal mechanism
involving the organic matrix.
1. Booster mechanism
2. Seeding mechanism

31. Booster mechanism
• Due to the concentration/action of the
enzymes, the concentration of the calcium
and phosphate ion which are building
stones of mineralization increases to such
a level that would lead to their
precipitation.

32. • Two theories
1. Robinson’s phosphatase theory
2. Cartier’s theory

33. Robinson’s phosphatase theory
• Alkaline phosphatase
organic phosphate inorganic phosphate
(local increase in phosphate ions)
+
calcium
hydroxyapetite crystals amorphous calcium phosphate

34. • Theory based on experiment on
alkaline phosphatase.
1. Calcifying cartilage contains more alkaline
phosphatase than non calcifying cartilage
2. When slices of cartilage removed from bone of
rachitic animals were incubated with calcium
and organic phosphates, hydroxyapetite
crystals were formed.

35. • Drawbacks:
1. Rachitic bone is an abnormal tissue
2. Alkaline phosphatase is observed in different
tissues which donot calcify
3. Inhibitors of certain other enzymes which
donot inhibit alkaline phosphatase activity are
found to be preventing mineralization

36. 4. Presence of inorganic phosphate and
calcium is not sufficient to induce
mineralization. Requires action of some
other enzymes.
5. The organic phosphate present in tissue
fluid is insufficient to induce
mineralization.

37. Alkaline phosphatase
• Is a group of enzymes that can cleave
phsphate ions from the organic phosphates
at an alkaline ph.
• It is found in cell membrane of hard tissue
forming cells and inorganic matrix of
calcifying tissues.

38. • Functions:
1. Hydrolizing organic phosphates to
provide inorganic phosphate ions
required for mineralization.
2. When associated with cell membranes,
play some role in ion transport.
Hideo Orimo. The mechanism of mineralization and the role of alkaline phosphatase in
health and disease. J Nippon Med Sch. 2010;77(1):4-12.

39. 3. Extracellular
activity;
help in crystal
growth by
hydrolyzing
pyrophosphate
(crystal poison)
The role of alkaline phosphatase in mineralization.Ellis E. Golub and
Kathleen Boesze-Battaglia. Basic science; 444-448

40. Cartier’s teory
• There are 2 substances which inhibit and
one which induce the process
• So, with proper control of their
concentration the mineralization takes
place

41. • Inducer:- adenosine
triphosphate
• Inhibitor:-
pyrophosphates

42. Seeding mechanism
• It refers to a presence of seeding or
nucleating substance which acts as a
mould/template on which the crystals are
deposited.
• Seeding substances
collagen
lipids
phosphoprotein
Protein polysaccharides

43. Collagen seeding theory/ nucleation
theory/collagen template theory
• Collagen- most important
seed in mineralization.
• Aminoacid residue with
charged side chains
provide a specific, spatial
arrangement that
constitute a template
matching for
hydroxyapetite.

44. • The growth of crystals round a nucleus or
seeding site is known as epitaxy .

45. Bind to template to form
hydroxyapetite crystals
Further growth of hydroxy apatite
crystals

46. • Specific ion binding sites:
1. For calcium - carboxyl associated with
asparatic and glutamic acid
residue
2. For phosphate – lysine and hydroxylysine

47. • Only the collagen with
64nm periodic
binding with three
dimensional
organization of
collagen
macromolecule has
the capability of
functioning as a seed.

48. • It is also suggested
that for seeding to
occurs within the
fibre, the phosphate
and calcium ions
must pass through the
gaps between the
tropocollagen
molecules.

49. Gaps filled with proteoglycans which bind
to calcium.
Calcium released by enzymatic
degradation of proteoglycans.
Removal of proteoglycans
attachment of phosphoproteins to collagen

50. break down by alkaline phosphatase
phosphate ion calcium ion
apetite crystals

52. • This theory is unable to explain
mineralization in all tissues.
Ex. Enamel is a mineralized tissue, but does
not contain collagen.
mineralization of cartilage begins in
ground substance and not in association of
collagen.

53. • Why doesnot initiate mineralization in all
connective tissue?

54. • Collagen in C.T that does not calcify, may
have spatial arrangement of charges
therefore unable to act as a suitable
template.

55. • In collagen of soft tissues, the charged site
could be protected by some ground
substance components which prevent the
attachment of the ions to initiate
mineralization.
• These substances are called crystal
poison.

56. • Collagen exhibits intrafibrillar pores
through which the calcium and phosphate
ion should pass through to reach the
nucleating sites located inside the fibrils.
• The gap between tropocollagen molecules
in calcified tissues – 0.6nm
in soft tissues – o.3nm

57. Other nucleating substances
• Lipids
 Phospholipids can act as a seed or a template
 Also capable of stabilizing amorphous calcium
phosphate which will later be transformed into
hydroxyapatite crystals.
 Also found in matrix vesicle

58. • Protein polysaccharides:
 proteoglycans and glycoseaminoglycans have
the capability of binding to calcium ions.
 Probably regulate the rate of mineralization
rather than initiation.

59. Control of mineralization by
cells
• All the components of supportive connective tissue,
including the matrix of fibres and ground substance and
its mineralisation with apatite, are the result of cell
activity.
• This activity of bone forming and bone removing cells is
clearly influenced by
hormones such a parathyroid, calcitonin and growth
hormone.
local chemical mediators --- growth factors and
interleukins.

60. Adele L. Boskey. Mineralization of bone and teeth. Elements. Vol 3;387-393.

61. Matrix vesicle theory
• Matrix vesicle:
extracellular, membrane-invested vesicle,
50-200nm in diameter,
formed by polarized budding from the
surface membrane of chondrocytes,
osteoblasts and odontoblasts.

63. • Induces precipitation of hydroxyapatite
crystals in vitro from solution containing
calcium and phosphate ions
• Also capable of crystal formation
• Suggest that the matrix vesicles have a
capacity to initiate mineralization.

64. • Found in:
hypertropic cartilage
bone
mantle dentin
fish scales
• Not found in:
enamel
circumpulpal dentin

65. • Two types:
• Type I:
round or ovoid in shape resembling
lysosomes
contain enzymes- acid phosphatase and
aryl phosphatase
can break down proteoglycans and
glycoseaminoglycans (inhibitors)

66. • Type II:
irregular membrane bound structures
enzymes – ATPase, alkaline phosphatase,
pyrophosphate, proteoglycan,
metalloproteinases, annexins
A2(II), A5 (V) and A6 (VI)
and calbindin

67. • Relatively less acid phosphatase
• Are also rich in phospholipids with great
affinity for calcium

68. Role of matrix vesicle
• Mineralization occurs in two steps.
1. Formation of hydroxyapatite crystals
within matrix vesicles
2. Propagation of hydroxyapatite through
the membrane into the extracellular
matrix

69. • By being extremely rich in alkaline
phosphatase activity,
vesicles hydrolyze organic phosphate substrates,
increases local availability of free phosphate ions
Bind to calcium ions
initiate apatite crystallization

70. • Such enzymatic
activity may also
remove putative
inhibitors of
mineralization,
including
pyrophosphate.

71. • phosphatidylserine, not only binds calcium, but
inorganic phosphate as well;
• hence an acidic phospho-lipid-calcium-
phosphate ( APL-Ca-P ) complex is formed.
• APL-Ca-P complexes appear to be unique to
mineralising tissues.

72. • The Ca+ are drawn into
the vesicle by a
membrane protein,
Annexin-V.
• It enables intraluminal
crystal growth.
• Binds directly to type II
and type X collagen
which may be important
for anchoring the vesicles
to the fibrous
components of the
matrix.
Balcerkaz et al. The roles of annexins and alkaline phosphatase in mineralization
process. Acta Biochimica Polonica. 2003;50(4):1019-1038.

73. Hideo Orimo. The mechanism of mineralization and the role of alkaline
phosphatase in health and disease. J Nippon Med Sch. 2010;77(1):4-12.

74. • Thus first crystal is
formed in the matrix
vesicle.
• Crystal growth
continues by further
addition of ions
• Rupture of vesicle
membrane

75. • Crystals released into
organic matrix
• Grow by using ions in
tissue fluids and
• mineralization spread
to surrounding matrix

76. • Mineralization progresses in the form of
spherical or calcospheric masses
• Which fuses with each other forming
uniformly mineralized matrix.

77. Enamel
• Calcification of enamel differs from the
above mentioned
• Mineralization and matrix formation occur
alongside enamel development
• Mineral content of enamel is 95-97% with
only a trace of organic matrix

78. • Enamel development begins with the
differentiation of cells of the oral epithelium
• Thickens to form a protruded inner enamel
epithelium
• Results in formation of ameloblasts which
secretes enamel proteins such as amelogenin
• Also involved in transport of calcium and
phosphate in enamel matrix

79. • Enamel proteins such as amelogenin
mediate the formation of hydroxyapatite
crystals from calcium and phosphate
through enamel biomineralization

80. • It occurs in two stages
1. Immediate partial mineralization
occurs in the matrix segments and
interprismatic substances
2. Maturation
MINERALIZATION OF
ENAMEL

81. • No matrix vesicles
• Nucleating substance– apetite crystals of dentin
• Tuftelin an acidic enamel protein localized to the
DEJ - participate in the nucleation of enamel
crystals.
• Other enamel proteins regulate enamel
mineralization by binding to specific surfaces of
the crystal and inhibiting further deposition.

83. 2. The second stage, or maturation, is characterized
by the gradual completion of mineralization .
• The process of maturation starts from the height
of the crown and progresses cervically. However,
at each level, maturation seems to begin at the
dentinal end of the rods.
• Thus there is an integration of two processes:
each rod matures from the depth to the surface,
and the sequence of maturing rods is from cusps
or incisal edge toward the cervical line

84. • Maturation begins before
the matrix has reached
its full thickness.
• The advancing front is at
first parallel to the
dentinoenamel junction
and later to the outer
enamel surface.
• The incisal and occlusal
regions reach maturity
ahead of the cervical
regions .

85. • The rate of formation of enamel is 4um/day, therefore to
form a layer of enamel of 1 mm thickness it would take
about 40 days.
• The crystal sizes increase further after tooth eruption due
to ionic exchange with saliva:
• Concomitantly the organic matrix gradually becomes
thinned and more widely spaced to make room for the
growing crystals.

86. • Amelogenesis is unique in many ways.
1. The secretory cell is an epithelial cell whereas all other
secretory cells of hard tissues are ectomesenchymal.
2. Noncollagenous proteins are involved in mineralization
of enamel whereas in all other hard tissues collagen
plays an important role.
3. The matrix of enamel does not contain collagen; in
other hard tissues collagen is the major protein.

87. 4. The matrix of enamel is partially mineralized; in other
hard tissues the matrix is nonmineralized. Enamel
therefore lacks distinct organic phase like osteoid,
predentin or cementoid.
5. There is no absorption of secreted matrix in other hard
tissues but in enamel formation 90% of secreted matrix
is absorbed and this activity is done by ameloblasts
itself.
6. After formation of enamel, ameloblasts undergo
apoptosis; hence enamel formation does not occur later
on. In other hard tissues formation occurs throughout
life.

88. • MINERALIZATION Begins once matrix is about
5µ thick.
• initiated by small crystallites within MatrixVesicles,
budded from odontoblasts.
Mineralization of dentin

89. Various Matrix Proteins Influence Mineralization:
• Gla-proteins, Phospholipids- Act as nucleators to
concentrate calcium.
• DPP- Binds to Ca, Controls Growth of H.A Crystals
• Osteonectin- Inhibits growth of H.A crystals,
promotes their Binding to Collagen
• Proteoglycans- inhibit premature mineralization seen
in predentin.

90. • MATRIX VESICLES contain Alkaline Phosphatase -↑
concentration of phosphates → combine with Calcium
→Hydroxyapatite Crystals.
• Crystals- grow rapidly, rupture the matrix vesicles
• Spread -clusters of crystallites → fuse with adjacent
clusters to form a continuous layer of mineralized matrix
• Initially- on the surface of the collagen fibrils and ground
substance, later within the fibrils- aligned with collagen.

91. • GLOBULAR(CALCOSPHERIC) :Deposition of HA
crystals in several discrete areas of matrix at any one time.
• Continued crystal growth → globular masses → enlarge
→ fuse → single layer of calcified mass.
• MANTLE DENTIN- matrix vesicles.
• LINEAR : When the rate of Dentin formation occurs Slowly -
Mineralization front appears more Uniform –
CIRCUMPULPAL DENTIN
Pattern of mineralization

92. INTER GLOBULAR DENTIN
RADIAL CRYSTAL GROWTH
GLOBULAR PATTERN
LINEAR PATTERN

93. • Nucleators – Gla proteins osteocalcin &
osteonectin
• BSP & Alkaline phosphatase – promote
mineralization
• Osteopontin – regulate growth of apetite
crystals
Mineralization of cementum

94. • Insulin like growth factor- present in
developing matured cementum
• Monitor mineralization
• Controls cell growth

95. • Uncalcified matrix – cementoid
• Proteoglycan located in unmineralized
cementum keratan sulfates-
lumican and fibromodulin.
• Calcium and phosphate ions present in
tissue fluids are deposited into the matrix
and are arranged as unit cell of
hydroxyapetite.

96. Adele L. Boskey. Mineralization of bone and teeth. Elements. Vol 3;387-393.

97. References
• Tencate’s oral histology. Antoni Nanci; 3rd edition.
• Orban’s oral histology. 5th edition.
• Essentials of oral biology. Maji Jose; 1st edition.
• Applied oral physiology.
• Biochemistry. U Satyanarayana; 1st edition.
• Text book of physiology. A.P.Krishna; 4th edition.
• The role of alkaline phosphatase in mineralization.Ellis E. Golub
and Kathleen Boesze-Battaglia. Basic science; 444-448
• Hideo Orimo. The mechanism of mineralization and the role of
alkaline phosphatase in health and disease. J Nippon Med Sch.
2010;77(1):4-12.