Download Biochemistry of bone

BONE METABOLISM
BONE MATRIX
Organic matrix - 20%
Inorganic matrix – 65%
Collagen
Hydroxyapatite – Ca10(PO4)6(OH)2
Glycoproteins
Octacalcium phosphate Ca8H2(PO4)6.5H2O
Bone specific
proteoglycans
Growth factors
Brusite – CaHPO4.2H2O
Amorphouse calcium phosphates –
Ca9(PO4)6
Water – 10%
BONE CELLS
Undifferentiated mesenchymal cells
Osteoblasts
Bone-lining cells
Osteocytes
Osteoclasts
Ionic composition of bone mineral
Cation
Ca2+
Mg2+
Na+
K+
value
6.66
0.18
0.32
0.02
(mmol/g of dry fat-free bone)
Anion
PO3CO32Citrate3Cl-
value
4.02
0.79
0.05
0.02
Schematic representation of physiological unit of bone
tissue
A = vascular space
B = osteocyte
C = bone canaliculi
D = gap junction between adjacent osteoblasts
E = connective tissue
Comparison of systemic ECF (plasma) with bone
ECF (electrolyte content, mmol/l)
Ca
Mg
K
Na
Pi
Cl
Plasma
1.5
0.7
4
140
1.8
100
Bone fluid
0.48
0.4
25
125
1.8
130
Bone-lining cells
Membrane which separates bone ECF and systemic ECF
Phases of bone formation




bone formation (embryonic development)
bone growth
maintenance of the skeleton – modeling and remodeling
healing of the skeleton (soft-tissue tissue trauma, fracture,
response to neoplasma, infection)
There is only one mechanism of bone formation and the
formation of bone follows the same sequence in
development, growth, and maintenance of skeleton:



within cartilage – enchondral ossification
within an organic matrix membrane – intramembranous
ossification
deposition of a new bone on existing bone – appositional
ossification
Diagramatic representation of the growth plate
Verical column of chondrocytes in reverse zone
(chondrocytes in different phases of the cell cycle).
Chondrocytes in the reverse zone are flat and
inactive, give rise to the daughter chondrocytes in
the proliferative zone).
In proliferative zone chondrocytes start to
proliferate.
In the zone of maturation chondrocytes start to
mature and give rise matrix vesicules, sites of
calcification.
In the upper hyperthrophic zone chondrocytes
start to enlarge and synthesize collagen type X, and
produce alkaline phosphatase. Matrix vesicules are
sites of the first mineral cristals.
Lower hyperthrofic zone containes degenerating
apoptotis condrocytes. Proliferating mineral crystals
spread in the extracellular matrix, small vessels
penetrate into the matrix, osteoblasts from bone
marrow deposit new bone and synthesize bone
extracellular matrix.
Matrix vesicle
Matrix vesicle is lying between 2
banded type II collagen fibrils.
Collagen type X, the proteoglycan
link protein and hyaluronic acid
binding region of proteoglycan are
attached to the matrix vesicles
surface.
The various phosphatases - alcaline
phosphatase, 5-AMPase, Ca-ATPase,
nucleoside triphosphatase
pyrophosphohydrolase (NTP-Pase)
and inorganic pyrophosphatase
(PPiase) are anchored in the
membrane.
In the sap – LDH, matrix vesicle
proteinases, carbonic anhydrase,
actin and various adhesive proteins
Components of the matrix vesicle
Alcaline phosphatase
• Hydrolysis of ester phosphate at the site of
mineralization to produce pyrophosphate (PP)
• Hydrolysis of local PP to two phosphates (Pi)
• Strong binding affinity to collagen
Carbonic anhydrase
• Regulates of pH inside of matrix vesicules
• Removes protons as biproduct of hydroxyapatite
formation
Annexins - Ca2+ ion channels
Annexin V (anchorin CII) and annexin II (calpactin)
Calcification of bone matrix is under cellular control
regulated by enzymes, proteins and phospoholipid
membrane:
Phase 1
1. Ca2+ attraction into the vesicle (Ca-binding phospholipids and Ca2+
chanels)
2. Accumulation of PO43- - action of alcaline phosphatase
3. Precipitation of calcium phosphate (noncrystaline amophous)
Phase 2
1. Matrix vesicle membrane breakdown (hydrolytic action of
phospholipases and proteinases)
2. Extravesicular hydroxyapatite crystal formation and proliferation
3. Itself sustaining process (constant homeostatic supplied level of Ca2+
and PO43-)
Biology of matrix vesicules
Matrix of hyperthrofic cartilage
contains typical cartilage
proteoglycans and collagen type
II.
During nucleation phase matrix
get to shrink, proteoglycans
aggregate. Ca2+ from adjacent
matrix withdrawn into
hyperthrophic cartilage matrix.
Chondrocytes and matrix vesicule
produce alkaline phosphatase
(production of free phosphates
for calcification).
Mineral crystals of hydroxyapatite
are formed and mineralization
continues.
Phases of enchondral ossification
•
•
•
•
•
•
hypertrophy of chondroblasts
calcification of cartilage extracellular matrix
penetration of blood capillaries
differentiation of osteoblasts and osteoclasts
osteoid formation
osteoid calcification
Remodeling of new bone
• bone resorption
• synthesis and secretion of the bone matrix
• calcification of newly formed bone lamellae
Bone resorption and bone formation are not
separated, independently regulated process.
Osteoblast and osteoclasts belong to structure, known as a
basic multicellular unit (BMU).
Osteoclasts are responsible to bone resorption
Proton pupm
pH 7
pH 4
Lysosomal enzymes
Acid phosphatase
Collagenase
Glycosidase
Sulphatase
Catepsins
Osteoblasts are responsible to organic
extracellular matrix formation.
Coupling
Sequential coordination between osteoclast-mediated resorption and
osteoblast-mediated formation (activation-resorption-formation- ARF)
In the young healthy adult resorption and formation are balanced such that
bone mass remains constant.
Pathological states developed when resorption and formation are unbalaced
(excessive bone loss or excessive bone resorption can occur).
Bone resoption and new formation in bone
remodeling unit (BMU )
Bone resoption and new formation in the BMU
http://www.roche.com/pages/facets/11/ostedefe.htm
Bone resoption and new formation in the BMU
Components which are responsible to
mineralization
Collagen type I
Bone glycoproteins :
bone sialoprotein, osteonectin, osteopontin
Bone phosphoproteins
Small proteoglycans
Growth factors
Hormones which regulate bone
metabolism
Systemic hormones of calcium homeostatic
system:
(normal physiological level of Ca2+ is 8.5 – 10.6 mg/100 ml)
Parathyroide hormone (PTH) – bone resorption (stimulation of
monocytes to transform to osteoclasts).
Calcitonine – bone formation (inhibition of monocyte to transform
to osteoclasts).
1,25-dihydroxycholecalciferol (1,25-(OH)2 vitamin D3) – bone
formation (activation of osteoblasts to synthesize collagen, promote
mineralization).
Calcium homeostasis
Parathyroid hormone
(parathyroid)
•
•
•
•
Released by low plasma calcium.
Stimulates bone resorption.
Prevents calcium excretion by kidneys.
Stimulates calcitriol synthesis.
Calcitriol (1,25-diOH-Vit. D)
(Vit. D in diet)
•
•
•
•
25-hydroxylation in liver
1-hydroxylation in kidney
Stimulates bone resorption.
Stimulates intestinal calcium absorption.
Calcitonin
(thyroid)
• Is released by high plasma calcium.
• Acts on bone osteoclasts to reduce
bone resorption.
• Net result of its action is a decline in
plasma calcium & phosphate.
Other systemic hormones :
Glucocortocuides – inhibition of bone formation.
Growth hormone (GH) – stimulation of bone formation through
somatomedins (growth factors IGF-1 and IGF-2).
Insulin – stimulation of synthetic activity of osteoblasts.
Thyroid hormones – stimulation of osteoclasts, activation of bone
remodelation.
Estrogens – inhibition of bone resorption (inhibition of osteoclastic activity
through specific local factors).
Catecholamines – antagonists of calcitonin.
Prostaglandins – different classes of prostaglandins have different effect,
which is dependent on concentration (10-9 – 10-7 mol/l stimulates
synthesei of collagen, 10-6 inhibits collagen synthesis.
Local factors which regulate bone metabolism
Factors which stimulate osteoblasts differentiation :
Bone morphogenic factor (BMF)
Platelet-derived growth factor (PDGF)
Fibroblast growth factor (FGF)
Insulin-like growth factor 1 (IGF-1)
Transforming growth factor-b (TGF-b.
Factors which regulate osteoclasts:
Colony-stimulating factor (CSF) – suppression of osteoclasts
development.
Interferons (g-interferon) – inhibition of osteoclasts differentioation.
Interleukins (IL-1, IL-3, IL-6, IL-11) – stimulates osteoclasts
differentiation
Biochemical markers of bone formation
• Alkaline phosphatase
– isoenzyme of bone alkaline phosphatase activity in
serum reflects osteoblastic activity.
• Osteocalcin - also called bone gammacarboxyglutamate (Gla) protein
– small, noncollagenous protein that is specific for bone
tissue and dentin
– its precise function remains unknown
– is synthesized predominantly by the osteoblast and is
mostly incorporated into the extracellular matrix of
the bone.
• Procollagen I extension propeptides
Biochemical markers of bone resorption
• Urinary calcium
– fasting urinary calcium test performed on a morning sample and
corrected for creatinine excretion (urinary calcium tests lack
sensitivity - reflects not only skeletal resorption, but also
intestinal absorption as well as renal tubular filtration and
reabsorption of calcium)
• Hydroxyproline
– is found mainly in collagen and represents about 13% of the
amino acid content of the molecule.
– Methods for measuring hydroxyproline - photometry, fluorometry
and high pressure liquid chromatography (HPLC).
• Urinary pyridinium crosslinks (pyridinoline and
hydroxypyridinoline).
Pyridinoline intramolecular cross-links
Location of the two intramolecular cross-linking sites in
collagen type I.
In fibrils, one site links N-telopeptide to helix and the other, C-telopeptide.
Pyridinoline cross-links are trivalent, joining two telopeptides to one tripledomain.