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

Pathologic Calcification

Mineralized Tissues

Tissue Inorganic Organic Bone _{Hydroxyapatite}

Fluorapatite Carbonated

Apatite

Collagen Type I

Cartilage Collagen Type II Chondroitin Sulfate

Tooth Collagen Type I

Mineralized Tissues

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

Organic

Parts

Calcium Phosphate Compounds

CaP Compound Formula

1. Amorphous Calcium Phosphate

2. Monocalcium Phosphate Anhydrous

3. Dicalcium Phosphate Anhydrous

4. Dicalcium Phosphate Dihydrate

5. Tricalcium Phosphate

Ca₃(PO₄)₂._3H

Calcium Phosphate Compounds

CaP Compound Formula

6. Tetracalcium Phosphate 7. Octacalcium Phosphate

Pentahydrate

8. Hydroxyapatite 9. Fluorapatite

10. Carbonated Apatite

Ca₄O(PO₄)₂

Amorphous Calcium Phosphate

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

– Bone

– Normal Dentition (Enamel, Dentin, and Cementum)

Amorphous Calcium Phosphate

• ACP forms instantaneously during the spontaneous precipitation from

supersaturated basic Ca2+_{and HPO} 4

2-aqueous solutions.

• ACP is generally viewed as a precursor to Hydroxyapatite (HAP) formation.

Hydroxyapatite

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

• Requirements for HAP formation are

– Calcium

– Enzyme : Carbonic anhydrase – Phosphate

Hydroxyapatite Formation

• Calcium (Unionized)

– Calcium Citrate

– Calcium Binding Protein

• Enzyme Carbonic anhydrase

Ca Salts

Phosphoprotein HPO₄

2-Hexose Monophosphate PO₄

3-Hexose Diphosphate H₂PO₄

-Glycerophosphate *Alkaline phosphatase

Hydroxyapatite Formation

• In plasma, Ca2+ _{and PO}

43- concentration

are supersaturated

Mechanisms of Hydroxyapatite Formation

• Homogeneous Nucleation (Booster Mechanism)

– More objections

• Heterogeneous Nucleation (Seeding Mechanism)

– Less objections

Homogeneous Nucleation

• Increase Ca2+_{and HPO}

42- concentration

• Alkaline phosphatase induce ions turn into the crystal nucleus

Ca2+

HPO₄

2-ALP

Heterogeneous Nucleation

• The promotion of the growth of one

crystalline substance on a “Seed”, different crystalline material having similar lattice spacing.

Ca2+ _{Hydroxyapatite}

HPO₄2- _Nucleus

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

Hydroxyapatite Hydroxyapatite Nucleus Crystal Growth

Heterogeneous Nucleation

• The growth of hydroxyapatite crystal

depend on the outer surface of the crystal that firmly bound H₂O.

• This surface called “Hydration Shell”

Hydration Shell Crystal Surface Crystal Interior

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 HPO}

42- deposit at the crystal

Heterogeneous Nucleation

• Other cations and anions such as Mg2+_,

Sr2+_{, F}-_{, CO}

32- 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

• Hydroxyapatite Substitution

Ca2+ _PO

• Seeds substances for heterogeneous nucleation

– Collagen Fiber – Chondroitin Sulfate – Phosphoproteins

– Lipids Most in_{abnormal condition} Less in

abnormal condition Most in

normal condition

Collagen Fiber as Seed

• There are 2 components of collagen act as seed for mineralization

1. End terminal of collagen helix

• COO-_{attract Ca}2+

• NH₃+_{attract HPO} 4

2-2. Phosphate bond to collagen

Collagen – Serine – OH ATP Collagen – Serine – OPO₃2- _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 HPO}

Phosphoproteins as Seed

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

Lipids as Seed

• 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.

Pathologic

Calcification

Collagen Fiber as Seed

Fibril Molecular

packing

Collagen molecule HZ

OZ

D D D D D

Cross striation (D = 65-67 n-m)

boundary Cross linke( )

located at hole zone

Important Inorganic Materials in

Mineralization

• Apatite --- Ca

₁₀

(PO

₄

)

₆

X

₂

–Hydroxyapatite ---

Ca

₁₀

(PO

₄

)

₆

(OH)

₂

–Fluorapatite ---

Ca

₁₀

(PO

₄

)

₆

F

₂

Hydroxyapatite

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

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

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

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

HAP vs FAP

• 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.

HAP – Critical pH = 5.5 FAP – Critical pH = 4.5

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

CPP – ACP

Clinical Correlation

• Dental Implant

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

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 Colony-Forming 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)

M-CSF +

Bone Remodeling

• 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.

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.

Osteoclast Active

Osteoblast

Bone Remodeling

• Bone remodeling can be divided into the 5 phases

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.

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)

*Require Carbonic anhydrase

Osteoclast action in Bone Resorption Osteoblast regulate Osteoclast action in Bone remodeling process

Bone Remodeling

• 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 materialwhich 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

Mechanical Factors and Bone Remodeling

Regulatory Factors in Bone Remodeling

5. Hormonal Factors

• 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

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 Factors

• 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)

Cytokines

• Interleukin (IL) • Prostaglandin (PG)

Growth Factors and Cytokines

Regulatory Factors

Stimulate Bone Formation

Stimulate Bone Resorption

Inhibit Bone Resorption

IFN-γ

Clinical Correlation

• Endodontics