fc

44

Mineralization

Introduction
Booster theory
Seeding theory
Matrix vesicle theory

Mineralization is the process of deposition of

minerals in the organic matrix, which is capable
of accepting the minerals. The process of
mineralization is an important step in formation
of hard tissue of the body.
The synthetic cells are responsible for
deposition of calcifiable organic matrix with
alkaliiie^phosphatase enzyme activity.
The mineral component of all hard tissues of
the body are chiefly calcium hydroxyapatite
crystals which is represented as Ca 10 (PO 4 )6
(OH)2. The biologic apatite crystal has the shape
of stubby rhombic prism which varies in size. The
crystallites of mesenchymal hard tissues are
approximately 100 x 200 x 50 x 50 A dimensions
where as hydroxyapatite of enamel forms a
considerably larger crystal which is 1,400A long
and 800A wide.
Although, tissue fluid contains calcium,
phosphate and other minerals, spontaneous

crystallization do not take place. This is probably

because of presence of substances inhibiting
crystal formation, requirement of energy for
mineralization and formation of unstable
insufficient amount of crystal, which is unable to
cause mineralization.
In this situation, mineralization can occur under
following circumstances:
1. If there is local increase in concentration of
minerals which allows formation of
sufficient ionic crystallites required for
mineralization. Such process that leads to
mineralization is called homogenous
nucleation.
2. In presence of a nucleating substance that
can act as a template for crystal formation
therefore decreasing the energy
requirement for mineralization. Nucleating

substance can initiate mineralization even

in the absence of increase in ionic
concentration. This is called heterogenous
mineralization.
3. The above mechanism will be effective if
there are means taremove the inhibitors of
mineralization.
Once crystal formation is initiated,

305

mineralization progresses rapidly, utilizing the

calcifying cartilage contains more alkaline
calcium and phosphate ions from the tissue fluid
phosphatase than non calcifying cartilage.
even at a low concentration of ionic content. Based 2. When slices of cartilage removed from
on above observations, theories have been put
bone of rachitic animals (affected by
forward to explain the process of mineralization.
richets) were incubated with calcium and
organic phosphates, hydroxyapatite crystals
1. Booster theory or Robinson's alkaline
were formed. From this experiment
phosphatase theory
Robinson came to a conclusion that rachitic
This theory is put forward by Robinson in 1923
bone contains alkaline phosphatase which
based on some of his experimental evidences.
is capable of splitting organic phosphate to
He proposed that, alkaline phosphatase enzymes
release
inorganic phosphates. This
present in the organic matrix of calcifying matrix
phosphate combines with calcium to produce
can hydrolyze organic phosphates such as
apatite crystals.
pyrophosphate or glucose 1-6 phosphate etc.
present in plasma and calcifying tissue fluid, and Robinson's theory of mineralization is not widely
release inorganic orthophosphate resulting in local accepted and is criticized for various reasons.
increase in phosphate ion concentration. This local 1. Robinson's studies were on rachitic bone
increase in ionic component has a boosting effect
which is an abnormal tissue. Whether the
which would increase the proportion of phosphate
result obtained in these studies can be
ions sufficient to cause spontaneous precipitation.
applied to normal bone is doubtful.
The phosphate ions combine with the calcium ions
available in tissue fluid to form hydroxyapatite 2. Alkaline phosphatase is observed in other
tissues which do not calcify.
crystals. According to him initially unstable
amorphous calcium phosphate is formed which 3. Inhibitors of certain other enzymes which
is then converted into stable calcium
do not inhibit alkaline phosphatase activity
hydroxyapatite.
are found to be preventing mineralization.
He has evolved his theory based on his 4. Experimental studies have shown that
experiments on alkaline phosphatase.
presence of inorganic phosphate and
calcium is not sufficient to induce
1. Robinson has observed in his studies that

pfQrai Biology

mineralization progresses rapidly, utilizing the

calcifying cartilage contains more alkaline
calcium and phosphate ions from the tissue fluid
phosphatase than non calcifying cartilage.
even at a low concentration of ionic content. Based 2. When slices of cartilage removed from
on above observations, theories have been put
bone of rachitic animals (affected by
forward to explain the process of mineralization.
richets) were incubated with calcium and
organic phosphates, hydroxyapatite crystals
1. Booster theory or Robinson's alkaline
were formed. From this experiment
phosphatase theory
Robinson came to a conclusion that rachitic
This theory is put forward by Robinson in 1923
bone contains alkaline phosphatase which
based on some of his experimental evidences.
is capable of splitting organic phosphate to
He proposed that, alkaline phosphatase enzymes
release inorganic phosphates. This
present in the organic matrix of calcifying matrix
phosphate combines with calcium to produce
can hydrolyze organic phosphates such as
apatite crystals.
pyrophosphate or glucose 1-6 phosphate etc.
present in plasma and calcifying tissue fluid, and Robinson's theory of mineralization is not widely
release inorganic orthophosphate resulting in local accepted and is criticized for various reasons.
increase in phosphate ion concentration. This local 1. Robinson's studies were on rachitic bone
increase in ionic component has a boosting effect
which is an abnormal tissue. Whether the
which would increase the proportion of phosphate
result obtained in these studies can be
ions sufficient to cause spontaneous precipitation.
applied to normal bone is doubtful.
The phosphate ions combine with the calcium ions
available in tissue fluid to form hydroxyapatite 2. Alkaline phosphatase is observed in other
tissues which do not calcify.
crystals. According to him initially unstable
amorphous calcium phosphate is formed which 3. Inhibitors of certain other enzymes which
is then converted into stable calcium
do not inhibit alkaline phosphatase activity
hydroxyapatite.
are found to be preventing mineralization.
He has evolved his theory based on his 4. Experimental studies have shown that
experiments on alkaline phosphatase.
presence of inorganic phosphate and
calcium is not sufficient to induce
1. Robinson has observed in his studies that

Alkaline Phosphatase

Organic Phosphate

Inorganic Phosphate
(Local increase in Phosphate Ions)

Calcium

Hydroxyapatite crystals -4

Amorphous Calcium Phosphate

Alkaline phosphatase and mineralization

mineralization. Rather this also requires

action of some other enzymes.
5. The organic phosphate present in tissue fluid
of calcifying matrix is insufficient to produce
sufficient inorganic phosphate ions to induce
mineralization.
Although this theory is been criticized by
various authors, the role of alkaline phosphatase
in mineralization can't be excluded.
Alkaline phosphatase is a group of enzymes
that can cleave phosphate ions from organic
phosphates at an alkaline pH. This enzyme is
found in cell membrane of hard tissue forming
cells and in organic matrix of calcifying tissue.
In addition to providing phosphate ions, alkaline
phosphatase may also be involved in ion transport
Neumann has proposed that alkaline phosphatase
may be playing important role in mineralization
by hydrolyzing pyrophosphate which is a known
crystal poison which prevents mineralization,
therefore helping in crystal growth.
The possible role of alkaline phosphatase in
mineralization can be:
1. Hydrolyzing organic phosphates to provide
inorganic phosphate ions required for
liberalization.
2. Ion transport
3. May help in removing crystal poisons.
2. Collagen seeding theory / nucleation
theory / collagen template theory

Some nndeating substances which have spatial

arrangement as that of riydroxyapatite crystals,
can act as a mould on template upon which
crystals can be laid down. Nucleating substance
can initiate nainerahzation even when the ionic
concentration is less and also reduce energy
required for mineralization.
Collagen is the most important seed playing a
significant role in mineralization. It is suggested
that certain aminoacid residues in collagen with

charged side chains, provide a specific, spatial

arrangement that constitute a template matching
for hydroxyapatite.
Calcium and phosphate ions present in the
extracellular fluid binds to these sites to form
hydroxyapatite crystals which grow further by
addition of ions. It has been observed that lysine
and hydroxylysine groups are specific ion binding
sites for phosphate ions, while carboxyl sites
associated with aspartic acid and glutamic acid
residues act as calcium binding sites to initiate
nucleation of hydroxyapatite crystals.
The role of collagen in mineralization was
suggested based on some experimental
evidences. When tendon collagen was added to
a solution containing calcium and phosphate,
crystal formation was found even when the
concentration of ions were lower than what is
required for spontaneous mineralization and it was
suggested that this had happened due to seeding
capacity of collagen. Only the collagen with 64nm
periodic banding with three dimensional
organization of collagen macromolecule has the
capability of functioning as a seed. The gaps
between the collagen molecules are filled with
proteoglycans which bind to calcium. Calcium is
released by enzymatic degradation of
proteoglycans. After the removal of
proteoglycans, phosphoproteins are attached to
the collagen which is broken down by alkaline
phosphatase to give rise to phosphate ion. These
ions combine to form apatite crystals in the gap
zone of collagen.
Support for this template theory can be
achieved from electron microscopic observation
of parallel arrangement of hydroxyapatite crystals
and collagen fibers.
This theory is'Unable to explain mineralization
in all tissued. For e.g.: enamel is a highly
mineralized tissue, but does not contain collagen.
Mineralization of cartilage begins in ground
substance and not in association with collagen.

Therefore possibility of other mechanism should

be considered.
Similarly another important question to be
answered is why collagen does not initiate
mineralization in all connective tissue. Possible
explanation for this includea. Collagen in connective tissue that does not
calcify, may have spatial arrangement of
charges that is different from the collagen
in calcifiable tissue therefore unable to act
as a suitable template.
b. 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. In calcifiable tissue these
substances may be removed by certain
mechanism leading to exposure of these
charged sites, followed by binding of ions
to initiate mineralization. Pyrophosphate is
a known crystal poison which is hydrolyzed
by alkaline phosphatase enzyme to expose
the binding sites.
c. Collagen exhibits mtraflbrillar pores through
which the calcium and phosphate ions
should pass through to reach the nucleating
sites located inside the fibrils. The gap
between tropocollagen molecules in
calcifiable tissues is 0.6nm which is large
enough to allow the passage of phosphate
ions which are of 0.4nm diameter. The gap
in soft tissue collagen is only 0.3nm through
which the phosphate ions cannot pass,
therefore cannot act as a template for
hydroxyapatite crystals.
Other nucleating materials
1. Lipids: Lipids have been identified as an
important factor associated with
mineralization process. Although the exact
role of lipids in mineralization is not

identified, experimental evidences suggest

that phospholipids can act as a seed or a
template for hydroxyapatite crystal
formation. Phospholipids are also capable
of stabilizing amorphous calcium phosphate
which will later be transformed into
hydroxyapatite crystals. Phospholipids are
also found in matrix vesicle, which can
participate in mineralization.
2. Protein polysaccharides: It has been
suggested by some investigators that protein
polysaccharides act as a seed for
mineralization. Experimental evidences
show that proteoglycans and
glycosaminoglycans have the capability of
binding to calcium ions. Probably these
protein polysaccharides regulate the rate of
mineralization rather than initiating
mineralization.
Matrix Vesicle Theory

Matrix vesicles are membrane bound vesicles

isolated from areas of calcification. These
structures bud off from the synthetic cells and
are released into the organic matrix. It has been
observed that the matrix vesicles induces
precipitation of hydroxyapatite crystals in vitro
from solutions containing calcium and phosphate
ions and also are capable of crystal formation
even when the solubility of product of calcium
and phosphate are as low as 2 millimoles2. The
above factors suggest that the matrix vesicles
have a capacity to initiate mineralization.
Two types of matrix vesicles have been
identified
Type I matrix vesicle are round or ovoid in shape
resembling lysosomes. They contain enzymes
such as acid phosphatase and aryl phosphatase.
These enzymes can break down proteoglycans
and glycosaminoglycans which are inhibitors of
mineralization.