Mineralisation PPT Dental Year 2 Semes

Supachai Chuenjitwongsa
Department of Biochemistry and Academic Affair
Faculty of Dentistry, CU

Mineralization and Calcification
• Mineralization
– Insoluble mineral salts bind to organic matrix

• Calcification
– Insoluble calcium salts bind to organic matrix

Pathologic Calcification

Mineralized Tissues
Tissue
Bone

Inorganic

Hydroxyapatite
Fluorapatite

Cartilage
Tooth

Carbonated
Apatite

Organic

Mineralized Tissues
• Human mineralized tissues are all have a
protein as their main organic constituent,
though the type of protein varies.

Collagen Type I

Collagen Type II
Chondroitin Sulfate

Collagen Type I

• The mineralized tissues all have a form of
Calcium Phosphate (CaP) as their main
inorganic constituent.

Calcium Phosphate Compounds
CaP Compound
1. Amorphous Calcium
Phosphate
2. Monocalcium Phosphate
Anhydrous
3. Dicalcium Phosphate
Anhydrous
4. Dicalcium Phosphate
Dihydrate
5. Tricalcium Phosphate

Organic
Parts

Calcium Phosphate Compounds
CaP Compound

Formula

Formula
Ca3(PO4)2.3H2O
Ca(H2PO4)2
CaHPO4
CaHPO4.2H2O
Ca3(PO4)2

Amorphous Calcium Phosphate
• ACP are significant relevance to Dentistry
because of their involvement in

6. Tetracalcium Phosphate
7. Octacalcium Phosphate
Pentahydrate

Ca4O(PO4)2
Ca8H2(PO4)6.5H2O

– Bone
– Normal Dentition (Enamel, Dentin, and
Cementum)

8. Hydroxyapatite
9. Fluorapatite

Ca10(PO4)6(OH)2
Ca10(PO4)6F2

– Dental Calculus (Pathologic Mineralization)
– Dental Caries (Demineralization)

10. Carbonated Apatite

Ca10(PO4)6CO3

Amorphous Calcium Phosphate
• ACP forms instantaneously during the
spontaneous precipitation from
supersaturated basic Ca2+ and HPO42aqueous solutions.
• ACP is generally viewed as a precursor to
Hydroxyapatite (HAP) formation.

Hydroxyapatite Formation

• Requirements for HAP formation are
– Calcium
– Enzyme : Carbonic anhydrase
– Phosphate

Hydroxyapatite Formation
Pyrophosphate

– Calcium Citrate
– Calcium Binding Protein

• Enzyme Carbonic anhydrase
H2O + CO2

• HAP is a stable form of CaP in neutral and
basic environment.

• Phosphate

• Calcium (Unionized)

Ca Salts

CA

Hydroxyapatite

H2CO3
Ca2+

Phosphoprotein

Hexose Monophosphate
Hexose Diphosphate
Glycerophosphate
*Alkaline phosphatase

ALP*

HPO42PO43H2PO4-

Hydroxyapatite Formation
• In plasma, Ca2+ and PO43- concentration
are supersaturated

Mechanisms of Hydroxyapatite Formation
• Homogeneous Nucleation
(Booster Mechanism)
– More objections

• Heterogeneous Nucleation
(Seeding Mechanism)

– Less objections

Homogeneous Nucleation
• Increase Ca2+ and HPO42- concentration
• Alkaline phosphatase induce ions turn into
the crystal nucleus
Ca2+
HPO42-

ALP

Heterogeneous Nucleation
• The promotion of the growth of one
crystalline substance on a “Seed”, different
crystalline material having similar lattice
spacing.
Ca2+
HPO42-

Seed

Hydroxyapatite
Nucleus

Heterogeneous Nucleation

Heterogeneous Nucleation

• Seeding mechanism promotes
mineralization by involving of ionic clusters
that act as nuclei formation, crystal growth
is presumed spontaneously until lack of
space for further growth

• The growth of hydroxyapatite crystal
depend on the outer surface of the crystal
that firmly bound H2O.
• This surface called “Hydration Shell”

Hydroxyapatite

Nucleus

Hydroxyapatite
Crystal Growth

Heterogeneous Nucleation
• Hydration shell has highly polarization that
attract both cations and anions into itself.
• The exchange reaction between crystal
surface and hydration shell occurred.
• Ca2+ and HPO42- deposit at the crystal
surface to grow the hydroxyapatite crystal.

Hydration Shell
Crystal Surface
Crystal Interior

Heterogeneous Nucleation
• Other cations and anions such as Mg2+,
Sr2+, F-, CO32- can be attracted by

hydration shell and deposit at the crystal
surface to form other type of apatite crystal
• Finally, these ions make impurities of the
hydroxyapatite crystal.

Heterogeneous Nucleation
• Seeds substances for heterogeneous
nucleation
– Collagen Fiber
– Chondroitin Sulfate
– Phosphoproteins

– Lipids

Most in
normal condition
Less in
abnormal condition

Most in
abnormal condition

Heterogeneous Nucleation
• Hydroxyapatite Substitution
Ca2+

PO43-

OH-

Na+
Mg2+
Sr2+

CO32HCO3HPO42-

FClCO32-

Collagen Fiber as Seed
• There are 2 components of collagen act as
seed for mineralization
1. End terminal of collagen helix
• COO- attract Ca2+
• NH3+ attract HPO42-

2. Phosphate bond to collagen
Collagen – Serine – OH
Collagen – Serine – OPO32*Phosphate attract Ca2+

ATP
ADP

Collagen Fiber as Seed
• Mineralization begin at hole zone of
collagen fiber.

Collagen Fiber as Seed (New Hypothesis)
• Polymer – Induced Liquid Precursor

Chondroitin Sulfate as Seed
• Highly negative charges of CS attracts
Ca2+ and then attract HPO42-.

Phosphoproteins as Seed

Lipids as Seed

• Phosphate group attached to serine of a
specific protein in mineralizing tissue, have
also been cited as seeds, especially in
dentin.

• Lipid materials were found in areas
undergoing early mineralizing of bone and
teeth (including enamel).
• Phosphatidyl Serine is principle lipid
component for seeding mechanism.
• Triglyceride, Free Fatty Acids and other
Phospholipids mainly act as seed in
pathologic conditions.

Collagen Fiber as Seed
• Mineralization begin at hole zone of
collagen fiber.

Pathologic
Calcification

Cross striation (D = 65-67 n-m)
Zone : classified by electron beam transmission
OZ HZ

D

D

D

D

D

Molecular
packing

Cross linke( )
located at
hole zone
boundary

Fibril

Collagen molecule

Important Inorganic Materials in
Mineralization
• Apatite --- Ca10(PO4)6X2

Hydroxyapatite
• A hexagonal network like a honeycomb
with channel extending right through the
structure.

–Hydroxyapatite --- Ca10(PO4)6(OH)2
–Fluorapatite --- Ca10(PO4)6F2

Hydroxyapatite
• Filling of these tunnels by Ca and anions
(OH, F) leads to characteristic adjustments that
best satisfy bond-length requirements.
• even slight changes in the ionic radii of the
tunnel atoms lead to expansion or contraction of
the tunnel. On this basis, it was surmised that
the ‘very critical fit’ of the fluorine and
hydroxyl ions was responsible for the greater
stability.

• The structure can be considered a tunnel
structure with walls composed of cornerconnected CaO6 and PO4 polyhedra as
relatively invariant units.

Fluorapatite
• By substituting for the OH- ion in the
apatite molecule during the development
phase of dentin and enamel, fluoride
fixes calcium, provides increased
stability to the mineral structure, and
promotes remineralization.

Fluorapatite
• Most of these substituents are harmless,
and sometimes necessary for the
organism, but some of them could be very
dangerous; they could be toxic or cause
irreversible modifications of FAP.

HAP vs FAP

HAP – Critical pH = 5.5

FAP – Critical pH = 4.5

• OH- arrangement is not in the same plane
as the Ca2+ triangle but it displace either
above or below the plane triangle.
Moreover, it’s direction can be upward or
downward in C – axis.
• These situation make hydroxyapatite
less regular and compact than
fluorapatite. Therefore, hydroxyapatite
is more readily dissolve in acid.

Clinical Correlation
• Preventive Dentistry
– Fluoridated Water
– Fluoride Tooth Paste
– Fluoride Mouthwash
– Fluoride Varnish
– Fluoride Gel

Clinical Correlation
• Operative and Restorative Dentistry
– Fluoride – Released Restorative Materials.
– Casein Phosphopeptide – Amorphous Calcium
Phosphate (CPP – ACP)

CPP – ACP

Clinical Correlation
• Dental Implant
– HAP – Coated Implant
“Osseointegration”.

Bone Cells
• Osteoprogenitor Cell
• Bone Forming Cells
– Osteoblast
– Osteocyte

• Bone Resorbing Cell
– Osteoclast

Osteoblastogenesis
• Osteoprogenitor cells develop from
Embryonic Mesenchymal Cell in bone
marrow.
• There are molecular signals activate
differentiation process that turn
osteoprogenitor cells to different types of
bone cells.

Osteoblastogenesis
1. Growth Factors and Genetics
– Regulation Genes
•
•

Ihh (Indian hedgehog)
Shh (Sonic hedgehog)

– Transcription Factors
•
•

Core – Binding Factor a-1 (Cbfa1)
Bone Morphogenetic Proteins (BMP)

Osteoblastogenesis
2. Differentiation Markers
–
–
–
–
–

Cbfa1
Osteopontin (OPN)
Alkaline Phosphatase (ALP)
Bone Sialoprotein (BSP)
Osteocalcin (OCN)

Osteoclastogenesis
• The osteoclasts originate from the bone
marrow hematopoietic stem cells known
as “Granulocyte – Macrophage ColonyForming Units” (GM-CFU), precursors of
macrophages and monocytes.

Osteoclastogenesis
• Regulating Molecules
– Macrophage Colony – Stimulating
Factor (M-CSF)
– Osteoprotegerin (OPG)
– Receptor Activator of Nuclear
Factor – κB (RANK)
– RANK Ligand (RANKL)

Bone Remodeling
M-CSF

+

• Bone is a dynamic tissue, in constant
resorption and formation, permitting the
maintenance of bone tissue, the repair of
damaged tissue and the homeostasis of the
phosphorus and calcium metabolism.
• Through this balanced phenomena, known as
the remodeling process, about 5% of cortical
bone and 20% of trabecular bone is renewed
per year.

Bone Remodeling
• Bone remodeling occurs throughout life, but
only up to the third decade is the balance
positive.
• It is precisely in the third decade when the
bone mass is at its maximum, this is
maintained with small variations until the age of
50. From then on resorption predominates and
the bone mass begins to decrease.

Bone Remodeling
• Bone remodeling can be divided into the 5
phases
– Quiescent
– Activation
– Resorption
– Formation
– Mineralization

Active
Osteoblast
Osteoclast

Bone Remodeling
• Quiescent
– Said of the bone when at rest. The factors
that initiate the remodeling process remain
unknown.

Bone Remodeling
• Activation
– Retraction of the bone lining cells
(elongated mature osteoblasts existing on the
endosteal surface)
– Digestion of the endosteal membrane by
Collagenase action.

*Require Carbonic anhydrase

Bone Remodeling
• Resorption
– Osteoclasts then begin to dissolve the
mineral matrix and decompose the osteoid
matrix.
– Process is completed by the macrophages
and permits the release of the growth factors
contained within the matrix
• Transforming Growth Factor Beta (TGF-β)
• Platelet Derived Growth Factor (PDGF)
• Insulin – Like Growth Factor I and II (IGF-I and II)

*

Osteoclast action in Bone Resorption

Bone Remodeling

Osteoblast regulate Osteoclast action
in Bone remodeling process

• Formation
– Preosteoblast grouping phenomena is produced,
attracted by the growth factors liberated from the
matrix which act as chemotactics and stimulate their
proliferation.
– Preosteoblasts synthesize a cementing substance
upon which the new tissue is attached, and express
bone morphogenetic proteins (BMP) responsible
for differentiation.
– Differentiated osteoblasts synthesize the osteoid
material which fills the perforated areas.

Bone Remodeling
• Mineralization
– Mineralization begins thirty days after
deposition of the osteoid, ending at 90 days in
the trabecular and at 130 days in the cortical
bone.

Regulatory Factors in Bone Remodeling
1. Genetic Factors --- transmit bone mass
2. Mechanical Factors --- depend on muscular
activities, rest, and weight
•

Related to mechanical properties of bone structure;
shear stress, strain, elastic modulus.

3. Vascular/Nerve Factors
•
•

Vascularization --- bone development
Innervation --- bone physiology

4. Nutritional Factors --- bone development and
mineralization

Mechanical Factors and Bone Remodeling

Regulatory Factors in Bone Remodeling
5. Hormonal Factors
•
•
•
•
•
•
•

Growth Factors

Regulatory Factors in Bone Remodeling
5. Hormonal Factors (Cont’)
•
•
•

Progesterone --- stimulate osteoclast
Insulin --- stimulate matrix synthesis
Glucocorticoids --- inhibit osteoclast

6. Local Factors
•
•
•

Growth Factors
Cytokines
Matrix Proteins

Growth Hormone --- stimulate collagen synthesis
and growth factors
Thyroid Hormone --- stimulate osteoid synthesis,
mineralization, and control resorption
Parathyroid Hormone --- stimulate resorption
Calcitonin --- inhibit resorption
Vitamin D --- regulate mineralization
Androgen --- stimulate osteoblast
Estrogen --- regulate osteoclastogenesis

•
•
•
•
•
•
•
•

Insulin – Like Growth Factor (IGF)
Transforming Growth Factor (TGF)
Bone Morphogenetic Protein (BMP)
Platelet – Derived Growth Factor (PDGF)
Fibroblastic Growth Factor (FGF)
Epidermal Growth Factor (EGF)
Macrophage Colony – Stimulating
Factor (M-CSF)
Tumor Necrosis Factor (TNF)

Cytokines
•
•

Interleukin (IL)
Prostaglandin (PG)

Growth Factors and Cytokines
Regulatory
Factors

Growth
Factors

Stimulate Bone
Formation
BMP-2
BMP-4
BMP-6
BMP-7

IGF-I
IGF-II
TGF-β
FGF
PDGF

Inhibit Bone Resorption
Cytokines

Clinical Correlation
• Periodontics
– Immunopathogenesis of Periodontal Diseases

IFN-γ
IL-4

Stimulate Bone
Resorption
TNF
FGF
PDGF

EGF
M-CSF
GM-CSF

IL-1
IL-6
IL-8
IL-11

PGE1
PGE2
PGG2
PGI2
PGH2

Clinical Correlation
• Endodontics
– Immunopathogenesis of Periapical Diseases