Bone Mineral Chemistry and Structure

JC Elliott, QMUL

1) General composition of bone2) Variability of composition of calcium

phosphate in mineralised tissues3) Identity of calcium phosphate in bone4) Explanation of variable composition-

structure of mineral5) Removal of mineral6) Formation of mineral

Importance of chemistry and structure of inorganiccomponent of bone

1) Bone mineral acts as store of essential ions2) It acts as a long term buffer against acids3) Can accumulate toxic metals4) To understand mechanical properties of bone5) To understand mineralisation and resorptionprocesses

Composition of Bone (wt % of whole tissue)

Inorganic 70Organic 22Water remainder

Inorganic is a calcium phosphateOrganic is mainly collagen

Composition of inorganic (wt % of whole tissue)

Ca 26PO4 34CO3 7Na 0.9Mg 0.7K 0.3H2O 3-6?

Composition is variablee.g. CO3 falls in metabolic acidosis

Size of crystals in bone

Much too small to see in light microscope!

Electron microscope shows they are mainlyplates approx 40nm long×20nm widevery thin

A Ca atom is about 1/5 nm across

Consequence of small size is crystals have largereactive surface area

Overall composition is deceptiveLook at anatomical and temporal variations

Changes in bone mineral with age of mineral

Young ----------------------------- Old

High CO3& acid phosphate Changes towardsCa10(PO4)6(OH)2

Low OH- & “crystallinity” Better crystallised

Identity of calcium phosphate in bones and teeth

Ca3(PO4)2 tricalcium orthophosphateCaHPO4 monetiteCaHPO4.2H2O brushiteCa8H2(PO4)6.5H2O octacalciumphosphateCa10(PO4)6(OH)2 hydroxyapatite

Chemical composition approximates toCa10(PO4)6(OH)2, hydroxyapatite

X-ray diffraction like hydroxyapatite

Composition of inorganic (wt % of whole tissue)Ca 26PO4 34CO3 7Na 0.9Mg 0.7K 0.3H2O 3-6?+…+

Composition is variablee.g. CO3 falls in metabolic acidosis

Inconsistent with hydroxyapatite

Need to look at structure of hydroxyapatite

Look at ball and spoke model

Size of ions in hydroxyapatite

Approximate PO43- ion as a sphere

Apatite is approximately hexagonalclose-packed spheres

Other ions (Ca2+, OH-) go is spaces between spheres

Result: apatite very stable and tolerant of differentsized ions: many ionic substitutions are possible

Substitutions in biological apatites

Ca2+ Sr2+, Pb2+, Na+, vacancyPO4

3- HPO42-, CO3

2-

OH- F-, Cl-, H2O, CO32-(limited)

Substitutions can be combined, butcharge balance must be maintained

Many ions can also be adsorbed on thelarge surface area of the bone crystals

In summary, variable composition is explained by:1) Replacing ions in the crystals2) Adsorbing ions on crystal surfaces

Resorption and mineralisation

Dissolution and precipitation of mineral

Resorption

Ca10(PO4)6(OH)2 + 2H+ --- 10Ca2+ + 6PO43- + 2H2O

acid

Mineralisation

Biomineralisation

Key to understanding the problem is that extracellular fluid is supersaturated with respect to hydroxyapatite

Problems:1) How do we nucleate the crystals?2) How do we stop the crystal growth?3) How is their orientation controlled?

Role of collagen

In the 1960’s, electron microscope studies showedthat the initial crystals of hydroxyapatite were associated with the cross-banding regions of collagenand X-ray diffraction showed that the hexagonal axis(long direction) of the crystals were parallel to the collagen fibres

Collagen continued

It was therefore suggested that collagen was responsiblefor the nucleation and orientation of the hydroxyapatitecrystals

Collagen continued(1970’s)

Big problem:Why does some collagen mineralise,whilst other identical collagen does not?

Epitaxial role of collagen has been subsequentlydeveloped by the finding that nucleation in vitro can be enhanced by certain peptides e.g. osteonectinOthers cause inhibition of crystal growth.

Molecules Associated with Calcifying Tissues

CollagenType I present in bone, dentine and cementum, but not enamel. Also present in many tissues that do not calcify.

Non-collagenous proteinsOccur in bone and dentine matrices and are mainly acidic phosphoproteins. The bind Ca and also bind to collagen. Might be a candidate for initiation of mineralisation when bound to collagen. This group also includes the gamma-carboxyglutamate-containing proteins (GLA proteins), osteonectin, osteopontin and bone sialoprotein.

Molecules associated with calcifying tissues (continued)

ProteoglycansThere is a clear association between proteoglycans and apatite crystals when bone mineralisation takes place in cartilage. However, the proteoglycan content in the cartilage falls during bone formation.

LipidsSince 1959, it is known that they occur at the developing front of mineralisation in bone. Their role is unclear.

Matrix vesiclesFirst described in calcifying cartilage in 1967. Also in bone, cementum and reparative dentine. About 100 nm in diam. When they bud off, the accumulate Ca and PO4. Calcification begins on the internal surface of the membrane, often with a single crystal inside. The vesicle increases in size and ruptures, releasing crystals.

Further reading:

Biomineralization, Principles and Concepts in BioinorganicMaterials Chemistry. Stephan Mann, Oxford Univ Press, 2001

On Biomineralization. HA Lowenstam and Stephen Weiner,Oxford Univ Press, 1989

Biomineralization, Chemical and Biochemical PerspectivesS Mann, J Webb and RJP Williams (eds)VCH Verlagsgesellschaft, Weinheim, Germany, 1989

Ca10(PO4)6(OH)2 + 2F- ----> Ca10(PO4)6F2

At higher F- ion concentrations:

Ca10(PO4)6(OH)2 + 20F- ----> 10CaF2 + 6PO43- + 2OH-

Ca10(PO4)6(OH)2

Ca10-x(HPO4)x(PO4)6-x(OH)2-x