BONE MATRIX

Tim Arnett

Department of Anatomy andDevelopmental Biology

University College London

Treated with bleach (hypochlorite) to digest collagen

leaves mineral component intact

Treated with hydrochloric acidto dissolve mineral

leaves collagen component intact

Collagen shrinkage on drying

Bone is made of mineral+ organic (mainly collagen)

components

Bone matrix

· bone is a composite material

· organic component is ~90% type I collagen- structural protein providing strength, flexibility

· remaining 10% of organic component is complex mixture of proteins, including growth factors, osteocalcin, osteonectin, osteopontin, glycoproteins; all produced by osteoblasts, along with type I collagen

Bone Matrix - 2

· organic matrix is mineralised after secretion

– unmineralised matrix = “osteoid”

· mineral component is hydroxyapatite – Ca10 (PO4)6 (OH)2

- tiny crystals impregnate & surround collagen fibres- provides rigidity, resistance to compression

· mineralisation of osteoid is dependent on the hormonally active form of vitamin D (1,25-dihydroxyvitamin D)

· vitamin D deficiency results in failure to mineralise– leads to rickets in children, osteomalacia in adults

Two types of bone

Woven (immature, fracture)

large, rounded osteocytes

osteocytes irregularly spaced

randomly oriented collagen fibres

variable collagen fibre diameter

rapid matrix mineralisation

forms rapidly

rapid turnover

Lamellar (mature, adult)

smaller, flattened osteocytes

osteocytes regularly spaced

collagen fibres show regular, “plywood” orientation – confers strength

regular collagen fibre diameter

delayed matrix mineralisation (few days)

forms slowly

slow turnover

Collagen

• stable, structural ECM protein

• collagen molecule = triple-stranded helix (3 �-chains)

• 25% of total body protein (mainly type I)

MAIN COLLAGEN TYPES…..

TYPE I: (90% of total): fibrils � fibres bone, skin, tendon, ligaments, cornea, internal organs

TYPE II: fibrils (form meshwork) not visible with light microscopecartilage, intervertebral disc, vitreous humour

TYPE III: fibrils → fibres (‘reticular’ - coarse mesh)skin, blood vessels, internal organs

TYPE IV: sheet-like networkbasal laminae

Collagen biosynthesis

Alberts et al – Molecular Biology of the Cell, Ed. 2

Amino acid composition of collagen chains

Alb

erts

et a

l–M

olec

ular

Bio

logy

of t

he C

ell,

Ed.

3

…Gly-X-Y…

X or Y frequently proline

Hydroxylated amino acids in collagen

Alberts et al – Molecular Biology of the Cell, Ed. 3

Pro-collagen

Alberts et al – Molecular Biology of the Cell, Ed. 3

“¼ stagger” model of collagen molecule alignment in fibrils

Alberts et al – Molecular Biology of the Cell, Ed. 3

Extracellular assembly of individual collagen molecules into fibrils- ‘quarter stagger’ model

Alberts et al – Molecular Biology of the Cell, Ed. 3

Covalent links between and within collagen molecules formed by action of lysyl oxidase on lysine and OH-lysine residues

Alberts et al – Molecular Biology of the Cell, Ed. 3

Non-collagenous proteins in bone

• ~ 10% of organic component of bone matrix

• 4 main groups: cell-attachment proteins

gamma-carboxylated (gla) proteins

proteoglycans

growth factors & blood proteins

Non-collagenous proteins in bone

• Thrombospondin (450 kD) - influences cell attachment - strong Ca2+ bindingFibronectin (440 kD) - influences cell attachment & spreading; contains RGD sequence; binds collagen

• Osteopontin (50 kD) - phosphorylated; contains RGD sequence; influences cell attachment & spreading; strong Ca2+ binding; not bone-specificBone sialoprotein (50 kD) - also affects cell attachment; strong Ca2+ binding; bone-specific

• Osteonectin (SPARC) (35 kD) - most abundant NCP: ~2% total protein in developing bone; binds collagen & hydroxyapatite; promotes calcium phosphate deposition in vitro

• Osteocalcin (BGP) (6kD) - contains γ-carboxy glutamyl residues (Ca2+ binding); bone-specific protein; low affinity Ca2+ binding; synthesis stimulated by 1,25(OH)2 vitamin D. Matrix gla-protein (9kD) - not bone-specific

• Proteoglycan I (biglycan) (170 kD) - 2 chondroitin sulphate chains; mainly in developing bone, function unknownProteoglycan II (decorin) (120 kD) - 1 chondroitin sulphate chain; present in all bone; binds collagen, may modulate fibrillogenesis

• Blood proteins (~25% of NCP) - including α2HS glycoprotein… and growth factors, including IGF-I, IGF-II, BMPs, PDGF, FGFs, TGFβ….

γ-carboxy-glutamic acid

H O

N C C

H CH2

CO O -

CO O -

Ca ++

γ-carboxylation is vit Kdependent process →

Alberts et al – Molecular Biology of the Cell, Ed. 3

Proteoglycans

Cartilage: aggrecan

Bone: decorin

Alberts et al – Molecular Biology of the Cell, Ed. 3

Targeted disruption of non-collagenous protein genesie, “knockouts”

Osteocalcin - increased osteoblast activity (bone formation) in knockout mice

Biglycan - (binds collagen + TGFβ) knockout mice show reduced growth rate + develop osteoporotic phenotype; double KO with decorin → more osteoporosis, premature osteoarthritis, abnormal collagen fibril formation

MGP - matrix γ-carboxyglutamic acid (gla) protein knockout mouse exhibits extensive, lethal calcification + cartilaginous metaplasia of media of all elastic arteries

Osteopontin - knockout mice show altered wound healing; resistance to osteoporotic effects of ovariectomy; reduced tumour metastasis; reduced inflammatory arthritis…

Naturally occurring ‘knockouts’

Osteogenesis imperfecta

• diverse family of genetic disorders

• mutations of α1 and α2 chains of type I collagen

Severity varies greatly, depending on the type of mutation, from prenatal lethality through impaired stature / tooth development and deformity to minimal deformity, sometimes with hearing loss or blue sclerae