MINERALIZATION

Introduction
▪ Mineralization: Deposition of mineral salts in and around the organic

matrix to make it a calcified structure or Process of deposition of

insoluble calcium salt in the tissue

▪ Important step in formation of Enamel, Dentin, cementum and bone

▪ 3 tissues- Dentin, cementum and bone– similar -Type I collagen

▪ Synthetic cells– secrete protein- creating suitable microenvironments +

stabilize calcium and phosphate ions – deposited -organic matrix

 Organic Matrix in Hard tissues

■ Organic Matrix- attract and organize calcium and phosphate ions into a structured

mineral phase– form of HA

■ Formative blast cells of calcified tissues produce -organic matrix constituents that

interact with the mineral phase.

✓ Dentin, Cementum and Bone – Type I Collagen, Non collagenous proteins

✓ Enamel- Amelogenin, Ameloblastin, Enamelin

 Mineral
■ Inorganic component of mineralized hard tissues- Calcium Hydroxyapatite crystal
(HA) Ca10(P04)6(0H)2 (Preceded by unstable amorphous calcium phosphate)

■ Unit cell of biologic apatite

– Ca, P and−OH)- hexagonal- stacked together- form the lattice of a crystal

– Repetitions of this arrangement produces crystals of various sizes

■ Crystals - needlelike or plate like , Enamel- long, thin ribbons

■ Apatite Crystallite- 3 surfaces for ion exchange- crystal Interior, crystal surface,
hydration shell (layer of water)

■ Substitutions : Mg, F, Na, Cl

 Factors control mineralization

▪ Tissue fluid is supe rsaturated with Ca & P ions- but spontaneous

precipitation of calcium phosphate d o e s not take place

✓ Inhibitory macromolecules-inhibit crystal formation

✓ Unstable-initial cluster of ions to f o r m a lattice structure

✓ Energy barrier- needs to be over come

Factors inhibiting Mineralization
➢ Mg2+

➢ Albumin

➢ Act locally in bone matrix- Proteoglycans

➢ Pyrophosphates

➢ Nucleotides

➢ Citrates
Factors Promoting Mineralization
➢ Alkaline phosphatase

➢ Nucleating substance (Collagen, Sulfated glycosamin oglycans, Proteoglycans,

Phospholipids)
 Mechanism of Mineralization
Mineralization occurs by aggregation of ions to form crystals. This process is called

Nucleation

▪ Homogenous nucleation (Booster Mechanism)- Increase in inorganic ions- Formation

of ionic cluster and spontaneous crystal precipitation

▪ Heterogeneous nucleation (Seeding Mechanism)- Seeding or Nucleating substance

acts as template- lower energy barrier- crystallization (without need of increased

ions)

Mechanism exist- Inactivation of inhibitors ( Pyrophosphate deposit on surface and prevent

growth )- Once crystallite formed- supersaturated solution- continue crystal growth

THEORIES OF MINERALIZATION

1. Robinson's Alkaline Phosphatase theory/ Booster theory

2. Nucleation theory/ collagen Seeding theory/collagen template

theory

3 . M a t r i x vesicle t h e o r y
1. Robinson's Alkaline Phosphatase theory/
Booster theory
Robinson proposed - 1923
Increase of
Organic
Hydrolyzed by inorganic
phosphates in Release of
Alkaline Phosphate Ions
plasma and Phosphate Ions
Phosphatase and spontaneous
calcifying tissues
precipitation
Hydrolyses PPi

+
Not Accepted Calcium

Amorphous
Hydroxyapatite calcium
crystals phosphate
 Role of Alkaline Phosphatase
■ Group of enzymes that cleave the phosphate ions at organic substrates at alkaline pH

■ Present on cell membrane of hard tissue forming cells & in the organic matrix

■ Provide phosphate ions & also involved in transport of ions

■ Hydrolyzing inhibitors -pyrophosphate which prevents mineralization- hence helps

crystal growth
Drawbacks

▪ Alkaline phosphatases seen in tissues and cartilage which do not calcify

▪ Organic phosphate not sufficient to produce inorganic phosphate to initiate

calcification process
 2. Nucleation theory/ collagen Seeding
theory/collagen template theory
■ N e u ma n n a n d N e u ma n n (1953)

■ Based on the concept of seeding or epitaxy ( Nucleating substance resemble Apatite crystals –act

as template)

■ Organic matrix components facilitate precipitation by acting as heterogeneous nucleating agents

■ According to this th eo r y- nucleus is formed probably in relation to collagen,

effective in aggregating Calcium and Phosphate ions- H A crystals grow

spontaneously
■ Formation of collagen based calcified tissues- deposition of

apatite crystals are catalyzed by specific atomic groups present on

the surface, holes and pores of collagen fibrils

■ Type 1 collagen- 70-80% mineral is within collagen fibril, rest in

between spaces
Nucleation theory/ collagen Seeding
theory/collagen template theory
Nucleation theory/ collagen Seeding
theory/collagen template theory
Nucleation sites and their nucleating agents

1. Ground su b stan c e:Sulfated glycosamin oglycans, Proteoglycans

2. Collagen and associated tissues: Collagen fibrils, Phosphoproteins

including Osteonectin (Induce Apatite formation)

3. Mitochondria in Osteoblasts: Indirect role in mineralization- Storage

sites of Calcium & Phosp h ate in the f o r m of amorphous CaPo4-

released – support growth of crystals

 Drawbacks

▪ Fails to explain mineralization in enamel (no collagen) and cartilage

(Mineralization begins in ground substance)

▪ Fails to explain n o mineralization in connective tissue ( Possible explanation

collagen molecules in soft tissues are closely packed together -prevent phosphate

ion access to intrafibrillary nucleation sites or inhibition by pyrophosphate

 3. Matrix vesicle theory

■ Crystals have been found to be formed in association with matrix vesicles

■ Matrix vesicles are small membrane bound structures, 25 to 250 nm in

diameter, lying free in the matrix, where calcification is known to be

underway

■ Rounded outgrowths of cell membrane that bud from osteoblasts,

chondrocytes and odontoblasts.

Matrix vesicles
Two types

■ Type I: round or ovoid ■ Type II: irregular

■ Containing lysosomes ■ Enzymes such as ATPase, alkaline

phosphatase, pyrophosphatase,
■ Enzymes such as acid phosphatase
proteoglycans, metalloproteinases but
and aryl phosphatase
relatively less acid phosphatase
■ Enzymes breakdown proteoglycans
■ Contain Phospholipids- great affinity
and glycosaminoglycans- which are
for calcium.
inhibitors of mineralization
Matrix vesicle theory
Enamel mineralization
➢ Matrix vesicles and Collagen are absent in enamel

➢ Enamel mineralization is thought to be achieved by crystal growth from already mineralized

dentin - by matrix proteins secreted by Ameloblasts

➢ Enamel proteins are of 2 types:-

1. 90% amelogenins- Proline, Histidine, Glutamine- helps to maintain space between
crystals.
2. 10% non-amelogenins- Tuftelin, enamelin, Ameloblastin -helps in nucleation and growth
of crystals.
Mineralization of Enamel and Dentin

Enamel Dentin
■ Formative cell- Ameloblasts ■ Formative cell- odontoblasts
■ Organic Matrix- Amelogenins, Non-
amelogenins ■ Organic Matrix- Collagen and non

■ HA> 90% collagenous proteins

■ Nucleation- Amelogenins? ■ HA- 67%

Non-amelogenins?
Dentin? ( Apatite crystals ■ Nucleation – Matrix vesicles
laid over enamel)
■ Prematrix- present-thick
■ Prematrix – None
■ Growth- Appositional ■ Growth- Appositional
Mineralization of Enamel and Dentin
Dentin
Enamel
■ Amelogenins ■ Dentin phosphoprotein-
Maintain space between crystals Highly anionic- binds to Ca+ -
Regulate crystal growth Transport to mineralization font-
control growth of crystals
Absence-hypoplastic teeth
■ Osteonectin
Inhibits growth of apatite crystals-
■ Non-amelogenins
But promotes binding – collagen
– Ameloblastin and Enamelin-
Nucleation and growth of ■ Gla-proteins
crystals Act as seed or nucleators- attract
– Tuftein- cell signaling calcium
Mineralization of Cementum and Bone

Cementum Bone
■ Formative cell- Cementoblasts ■ Formative cell- osteoblasts

■ Organic Matrix- Collagen and non ■ Organic Matrix- Collagen and non
collagenous proteins
collagenous proteins
■ HA- 50-60%
■ HA- 45-50%
■ Nucleation – Matrix vesicles
■ Nucleation- Matrix vesicles
■ Prematrix- present during formative
■ Prematrix – Present-thin phase- thin

■ Growth- Appositional ■ Growth- Appositional

Thank you