Structure, Growth, and Structure, Growth, and Mineralization of BoneMineralization of Bone

Lecture 4Lecture 4

Case study:Case study:

A young man was of average height A young man was of average height till he was 15 years of age, but he till he was 15 years of age, but he was still growing at 24 years. He was was still growing at 24 years. He was 6ft. 7inches tall, but his bone were 6ft. 7inches tall, but his bone were weak. What do you think was the weak. What do you think was the problem?problem?

Bone is a specialized connective tissue that, Bone is a specialized connective tissue that, along with cartilage, forms the skeletal along with cartilage, forms the skeletal systemsystem

The role of boneThe role of bone Bone has a protective and supportive role. Bone has a protective and supportive role. It is metabolically active, and stores many It is metabolically active, and stores many

mineralsminerals It provide defense against acidosis It provide defense against acidosis It forms a trap for some dangerous minerals It forms a trap for some dangerous minerals

such as leadsuch as lead

BoneBone

A.Compact (cortical) Not completely solid, but permeated by Haversian canals containing blood vessels and bone cells.

B.Cancellous (trabecular) Honey comb network of calcified tissue on the inner side.

The terms cortical and cancellous describe the gross arrangement of bone tissue. In general bones are compact on the outside and cancellous (spongy) on the inside.

Microscopically bone is composed of cells and extracellular matrix

Since the surface to volume ratio of Since the surface to volume ratio of cancellous bone is so large it has an cancellous bone is so large it has an important role to important role to play in play in calcium ion calcium ion homeostasishomeostasis (the importance of (the importance of cancellous bone in Calcium cancellous bone in Calcium homeostasis is exemplified by the fact homeostasis is exemplified by the fact that that 99% of the total body Calcium is 99% of the total body Calcium is stored within the skeletonstored within the skeleton).).

Figure 1. Structure of bone

A scanning electron image (B) that shows bone collagen fibrils in A scanning electron image (B) that shows bone collagen fibrils in both longitudinal and cross sections. both longitudinal and cross sections.

The back-scattered electron image (C) shows the regular The back-scattered electron image (C) shows the regular patterns of collagen in layers in bone, which is why normal bone is patterns of collagen in layers in bone, which is why normal bone is

called lamellar bone.called lamellar bone. Images from Marian YoungImages from Marian Young

Figure 2. Cells in bone structure

Biochemical functions of bone Biochemical functions of bone cellscells

Osteoblasts:Osteoblasts:• Synthesize webs of collagen fibers, proteoglycans and Synthesize webs of collagen fibers, proteoglycans and

glycoproteins. glycoproteins. • Control bone mineralization.Control bone mineralization.

Osteocytes:Osteocytes: • May contribute to the maintenance of calcium homeostasis , May contribute to the maintenance of calcium homeostasis , • May have a role in activating bone turnover.May have a role in activating bone turnover.

Osteoclasts:Osteoclasts:• They act in bone resorption and the release of calcium and They act in bone resorption and the release of calcium and

phosphate into the plasma.phosphate into the plasma.• Osteoclasts are able to resorb bone by selectively producing an Osteoclasts are able to resorb bone by selectively producing an extremely low pH within the immediate micro-environment of extremely low pH within the immediate micro-environment of their action by carbonic anhydrase IItheir action by carbonic anhydrase II

Osteoprogenitor cellsOsteoprogenitor cells • These are functionally undifferentiated mesenchymal cellsThese are functionally undifferentiated mesenchymal cells• Under the appropriate stimulation these cells may Under the appropriate stimulation these cells may

differentiate into functional osteoblasts. differentiate into functional osteoblasts. Lining cellsLining cells

• Are Are former osteoblastsformer osteoblasts which have become flat and pancake- which have become flat and pancake-shaped, lining the entire surface of the bone. shaped, lining the entire surface of the bone.

• Are Are responsible for immediate release of calcium from the responsible for immediate release of calcium from the bone if the blood calcium is too low. bone if the blood calcium is too low.

• Protect the bone from chemicals in the bloodProtect the bone from chemicals in the blood which dissolve which dissolve crystals (such as pyrophosphate). crystals (such as pyrophosphate).

• Have receptorsHave receptors for hormones and factors that initiate bone for hormones and factors that initiate bone remodeling.remodeling.

Bone consists of Bone consists of mineral saltsmineral salts (mainly (mainly

calcium phosphate, approximately 60% of calcium phosphate, approximately 60% of

weight) deposited on an weight) deposited on an organic matrixorganic matrix.. Water comprises approximately 25% of Water comprises approximately 25% of

adult bone mass.adult bone mass.

The matrix, which is formed before the The matrix, which is formed before the mineral is deposited, and can be mineral is deposited, and can be considered the scaffolding for the boneconsidered the scaffolding for the bone

It consists of It consists of proteinsproteins, of which , of which collagen type I is the most important, collagen type I is the most important, proteoglycansproteoglycans, and a smaller fraction , and a smaller fraction of of lipids and waterlipids and water..

Bone matrix

Table 1: Bone matrix compositionTable 1: Bone matrix composition

Water and matrix (40% of weight)

1. Collagen •Type 1 (90% of protein content) •Other types

2. Proteoglycans Veriscan, Decorin, Biglycan

3. Glycoproteins Alkaline phosphtase, Fibronectin,Osteonectin, Osteopontin, Bone sialoprotein, Matrix extracellular protein

4. Gla-proteins Matrix gla-protein

5. Serum proteins

Figure 3: Main features of the collagen molecule and its relation to the structure of the fibril

OsteogenesisOsteogenesis ImperfectaImperfecta Osteogenesis imperfectaOsteogenesis imperfecta ( (OIOI and and

sometimes known as sometimes known as Brittle Bone Brittle Bone DiseaseDisease, or "Lobstein syndrome") is a , or "Lobstein syndrome") is a genetic bone disorder. People with OI are genetic bone disorder. People with OI are born without the proper born without the proper proteinprotein ( (collagencollagen), ), or the ability to make it, usually because or the ability to make it, usually because of a deficiency of of a deficiency of Type-I collagenType-I collagen. .

Bone matrix: non-collagen, calcium Bone matrix: non-collagen, calcium binding proteinsbinding proteins

They are negatively charged They are negatively charged glycoproteinsglycoproteins

Therefore, they have a high calcium Therefore, they have a high calcium binding potential and are implicated in binding potential and are implicated in bone calcificationbone calcification

They include Osteopontin, Osteonectin, They include Osteopontin, Osteonectin, Osteocalcin, Matrix extracellular protein, Osteocalcin, Matrix extracellular protein, Bone sialoproteinBone sialoprotein

Osteopontin (RMM 32 600 kD):Osteopontin (RMM 32 600 kD): • Rich in aspartic acid + some sialic acid.Rich in aspartic acid + some sialic acid.• Synthesis is stimulated by vitamin D. Synthesis is stimulated by vitamin D. • Bind hydroxyapatite.Bind hydroxyapatite.• Associated with the attachment of osteoclasts to Associated with the attachment of osteoclasts to

the matrix. the matrix. • Binds to integrinsBinds to integrins• Increases angiogenesis (makes new blood vessels) Increases angiogenesis (makes new blood vessels)

which enhances bone resorption in some situationswhich enhances bone resorption in some situations

Osteonectin (RMM 35 000 kD):Osteonectin (RMM 35 000 kD):• Binds collagen and hydroxyapatite.Binds collagen and hydroxyapatite.• Also referred to as "Bone connector“, and may Also referred to as "Bone connector“, and may

regulate mineralizationregulate mineralization • Its synthesis is associated with bone formation Its synthesis is associated with bone formation

and remodeling.and remodeling.

OsteocalcinOsteocalcin (MW 11 000)(MW 11 000)::• The smallest MW. The smallest MW. • Contains γ-carboxyglutamic acid (2 or 3 residues) Contains γ-carboxyglutamic acid (2 or 3 residues) • Binds to hydroxyapatite (may regulate crystal size)Binds to hydroxyapatite (may regulate crystal size)• Its synthesis is stimulated by vitamin D.Its synthesis is stimulated by vitamin D.• Vitamin K is needed for the carboxylation of Vitamin K is needed for the carboxylation of

glutamic acid.glutamic acid.• Used as a marker of bone metabolism, as its Used as a marker of bone metabolism, as its

production and levels in the blood reflect production and levels in the blood reflect osteoblastic activity.osteoblastic activity.

Matrix Gla protein (MW 12 000):Matrix Gla protein (MW 12 000):• Contain γ-carboxyglutamic acid.Contain γ-carboxyglutamic acid.• Second smallest MW. Second smallest MW. • Synthesis is stimulated by vitamin D & require Synthesis is stimulated by vitamin D & require

vitamin K.vitamin K.• Function is not clear, but it appears to inhibit Function is not clear, but it appears to inhibit

mineralizationmineralization

Bone Sialoprotein:Bone Sialoprotein:• Rich in sialic acid.Rich in sialic acid.• Binds to integrins, may assist cancer cellsBinds to integrins, may assist cancer cells • No clear function.No clear function.

Fibronectin Fibronectin • Relatively abundantRelatively abundant• May help regulate osteoblast differentiationMay help regulate osteoblast differentiation

Types of bone matrixTypes of bone matrix Woven bone:Woven bone:

• Collagen fibrils are distributed within the matrix in a Collagen fibrils are distributed within the matrix in a haphazard arrangement. haphazard arrangement.

• It is rapidly formed however it is mechanically weak. It is rapidly formed however it is mechanically weak. • It is the first bone matrix formed during skeletal growth and It is the first bone matrix formed during skeletal growth and

development and healing. development and healing. • The presence of woven bone in the context of mature skeleton The presence of woven bone in the context of mature skeleton

is abnormal but non-specific. is abnormal but non-specific. Lamellar bone:Lamellar bone:

• Collagen fibrils have an ordered arrangement in “curving Collagen fibrils have an ordered arrangement in “curving linear arrays”. linear arrays”.

• This is a mechanically much more sound matrix. This is a mechanically much more sound matrix. • It is the type of bone found in the mature skeleton. It is the type of bone found in the mature skeleton.

Bone MineralsBone Minerals Bone mineral is so closely associated with the Bone mineral is so closely associated with the

organic matrix, especially with the collagen organic matrix, especially with the collagen component. It consists of:component. It consists of:

19-26% Calcium (Ca19-26% Calcium (Ca2+2+)) 9-12% Phosphate (Po9-12% Phosphate (Po44

3-3-))

2-4 % Carbonate (Co2-4 % Carbonate (Co332-2-))

0.2- 0.3 % Magnesium (Mg0.2- 0.3 % Magnesium (Mg2+2+)) Bone also contain Na and some other Bone also contain Na and some other

minerals in small amountsminerals in small amounts

The mineral is composed of narrow crystallites in the The mineral is composed of narrow crystallites in the

form of long cylinders; about 5 nm wide (60-70 nm in form of long cylinders; about 5 nm wide (60-70 nm in

length), arranged parallel to the collagen fibers. length), arranged parallel to the collagen fibers.

Some cylinders aggregate to from thicker Some cylinders aggregate to from thicker

structures approximately 20 nm wide. structures approximately 20 nm wide.

The crystal structure is similar to, but not identical with The crystal structure is similar to, but not identical with

naturally occurring hydroxyapatite [Canaturally occurring hydroxyapatite [Ca1010 (PO (PO44))66 (OH) (OH)22 ]. ].

Bone mineral has a higher content of carbonate and Bone mineral has a higher content of carbonate and

some phosphate may be replaced by sulphate or some phosphate may be replaced by sulphate or

silicates. silicates.

The crystal structure of hydroxyapatiteThe crystal structure of hydroxyapatite

BONE FORMATION AND GROWTHBONE FORMATION AND GROWTH

Bone Formation:Bone Formation: occurs by the coordinated activity of: occurs by the coordinated activity of: chondrocytes, osteoclasts and osteoblastschondrocytes, osteoclasts and osteoblasts

Undifferentiated mesenchymal cellsUndifferentiated mesenchymal cells Transforming growth factors (TGFs), other growth factors,

chondrogenic stimulating activity (CSA), steroid and peptide hormones and collagens & other extracellular matrix proteins

A- Chondrogenic lineA- Chondrogenic line ) ) cartilage formingcartilage forming((

Chondrocytes + osteoblastsChondrocytes + osteoblasts

B- Osteogenic line) bone forming(

Osteoblasts

A- Chondrogenic line (cartilage forming)A- Chondrogenic line (cartilage forming) Chondrocytes and osteoblastsChondrocytes and osteoblasts

• • A rod of Cartilage is formed by chondrocytes A rod of Cartilage is formed by chondrocytes • Osteoid tissue is formsd by osteoblasts outside the cartilage rodOsteoid tissue is formsd by osteoblasts outside the cartilage rod

• (Osteoid formation, Mineralization, Vascularization)10-1510-15

• Osteoblasts become embeded in osteoid & mature osteocytesOsteoblasts become embeded in osteoid & mature osteocytes

• Cartilage rod (center) Cartilage rod (center) bone marrow bone marrow

• Cartilage boneCartilage bone

Growth stops when all cartilage (except at the articular surface of Growth stops when all cartilage (except at the articular surface of the joint) is replaced by bone, and the epiphyses close.the joint) is replaced by bone, and the epiphyses close.

Bone formation by Endochondral ossification (cartilage is first formed and later replaced by bone)

)most of the skeleton (

OsteoblastsOsteoclasts

)Mineralization + vascularization(

Case study:Case study: A young man was of average height A young man was of average height

till he was 15 years of age, but he till he was 15 years of age, but he was still growing at 24 years. He was was still growing at 24 years. He was 6ft. 7inches tall, but his bone were 6ft. 7inches tall, but his bone were weak. What do you think was the weak. What do you think was the problem?problem?

Many factors play a role in controlling Many factors play a role in controlling growth of bone including certain growth of bone including certain hormones, local mediators and hormones, local mediators and nutritional statusnutritional status

Factors affecting formation & Factors affecting formation & growth of bonegrowth of bone

Pitutary gland:Pitutary gland:• Hypopituitarism → reduced rate of skeletal growthHypopituitarism → reduced rate of skeletal growth

Growth hormone:Growth hormone:• Important until epiphesial closure.Important until epiphesial closure.• Acts by: Acts by:

• influencing IGF-1 (regulate IGF-1 production) influencing IGF-1 (regulate IGF-1 production) • stimulation of cell division (prechondrocytes + stimulation of cell division (prechondrocytes +

osteoblasts)osteoblasts)

Thyroid hormone:Thyroid hormone:• May stimulate chondrocyte maturation.May stimulate chondrocyte maturation.

Sex hormones:Sex hormones:• Oestrogen help regulate the rates of bone Oestrogen help regulate the rates of bone

formation and bone resorption. formation and bone resorption.

• It inhibits osteoclasts and their precursors. It inhibits osteoclasts and their precursors. • It is also required for the process of epiphyseal It is also required for the process of epiphyseal

closure in both sexes and testosterone additionally closure in both sexes and testosterone additionally in males.in males.

GlucocorticoidsGlucocorticoids

Hormonal AbnormalitiesHormonal Abnormalities

Oversecretion of hGH during childhood Oversecretion of hGH during childhood produces giantismproduces giantism

Undersecretion of hGH or thyroid hormone Undersecretion of hGH or thyroid hormone during childhood produces short statureduring childhood produces short stature

Both men or women that lack oestrogen Both men or women that lack oestrogen receptors on cells, or are unable to convert receptors on cells, or are unable to convert testosterone into oestrogen grow taller than testosterone into oestrogen grow taller than normal (the case mentioned earlier)normal (the case mentioned earlier) oestrogen is responsible for closure of growth oestrogen is responsible for closure of growth

plateplate

Local mediators (regulators)Local mediators (regulators)

Bone cells produce molecules (usually proteins) that Bone cells produce molecules (usually proteins) that

communicate with other cells. communicate with other cells.

They act on nearby cells, and thus are considered They act on nearby cells, and thus are considered

local regulators. local regulators.

These factors control cell division (proliferation) These factors control cell division (proliferation)

One important pair are One important pair are Indian hedgehog Indian hedgehog and and PTHrpPTHrp

A mutation causing a constant activation of PTH A mutation causing a constant activation of PTH receptor causes short stature and other signs of receptor causes short stature and other signs of hyperparathyroidism, but with low serum level hyperparathyroidism, but with low serum level (Jansen’s chondrodysplasia)(Jansen’s chondrodysplasia)

Another mutation causing inactive receptor can be Another mutation causing inactive receptor can be fatal in the foetus, or the bones fail to elongate fatal in the foetus, or the bones fail to elongate (Blomstrand chondrodysplasia)(Blomstrand chondrodysplasia)

More information can be found at: More information can be found at: http://depts.washington.edu/bonebio/ASBMRed/ghttp://depts.washington.edu/bonebio/ASBMRed/growth.htmlrowth.html

Blomstrand Blomstrand chondrodysplasiachondrodysplasia

Jansen’s Jansen’s chondrodysplasiachondrodysplasia

Examples of Local mediators:Examples of Local mediators:

Example 1: fibroblast growth factors.Example 1: fibroblast growth factors.

A mutation affecting FGF Receptor-3 leads toA mutation affecting FGF Receptor-3 leads to

achondroplasiaachondroplasia, the commonest cause of human , the commonest cause of human

short statureshort stature..

Achondroplasia. This picture shows twin brothers .The twin on the right has achondroplasia.

Example 2: Transcription factors, controlling Example 2: Transcription factors, controlling the formation of mRNAs.the formation of mRNAs.

Mutations in TF msx1 & msx2 leads to abnormalities Mutations in TF msx1 & msx2 leads to abnormalities in in craniofacial craniofacial development.development.

Growth factorsGrowth factors

Bone morphogenetic proteins (BMPs):Bone morphogenetic proteins (BMPs): o BMPs are produced in the bone or bone marrow. BMPs are produced in the bone or bone marrow. o They bind to BMP receptors that are on They bind to BMP receptors that are on

mesenchymal stem cells within the bone marrow.mesenchymal stem cells within the bone marrow.o This causes the cells to produce Cbfa 1, which is This causes the cells to produce Cbfa 1, which is

a factor that activates the DNA so proteins can be a factor that activates the DNA so proteins can be made made

o When Cbfa 1 activates the genes, the cells When Cbfa 1 activates the genes, the cells differentiate into mature osteoblasts.differentiate into mature osteoblasts.

o Without Cbfa 1, the cells would turn into fat cells Without Cbfa 1, the cells would turn into fat cells instead.instead.

Insulin-like growth factors (IGFs):Insulin-like growth factors (IGFs): o These growth factors are produced by osteoblastic These growth factors are produced by osteoblastic

cells in response to several bone active hormones, cells in response to several bone active hormones, such as parathyroid hormone and estrogens, or such as parathyroid hormone and estrogens, or BMPs.BMPs.

o IGFs accumulate in the bone matrix and are IGFs accumulate in the bone matrix and are released during the process of bone remodeling released during the process of bone remodeling by osteoclasts.by osteoclasts.

o IGFs stimulate osteoblastic cell replication -- in IGFs stimulate osteoblastic cell replication -- in other words, they cause the osteoblasts to divide, other words, they cause the osteoblasts to divide, forming new cells. They may also induce forming new cells. They may also induce differentiation. differentiation.

CytokinesCytokines

Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor

necrosis factor (TNF) family of cytokinesnecrosis factor (TNF) family of cytokines

These factors are produced by osteoblastic cells in These factors are produced by osteoblastic cells in

response to systemic hormones or other cytokines. response to systemic hormones or other cytokines.

IL-6 can cause: IL-6 can cause: Bone marrow stem cells to differentiate into pre-osteoclasts Bone marrow stem cells to differentiate into pre-osteoclasts

Changes in proliferation and differentiation of osteoblasts Changes in proliferation and differentiation of osteoblasts

Inhibition of apoptosis of osteoblasts Inhibition of apoptosis of osteoblasts

Dietary Factors:Dietary Factors: Calcium, phosphate, vitamins D, A and C are Calcium, phosphate, vitamins D, A and C are

most important for endochondrial ossification.most important for endochondrial ossification. Also important (but less) are: zinc, magnesium Also important (but less) are: zinc, magnesium

and vitamin K.and vitamin K. More recently researchers investigated the effects More recently researchers investigated the effects

of moderate magnesium (Mg)-restricted diet on of moderate magnesium (Mg)-restricted diet on bone formation and bone resorption in rats. bone formation and bone resorption in rats. Mg-restricted diet induced a decrease in bone Mg-restricted diet induced a decrease in bone formation and an increase in bone resorption. formation and an increase in bone resorption.

Restriction of energy intake also effects bone Restriction of energy intake also effects bone formation. formation.

MINERLIZATION OF BONEMINERLIZATION OF BONE

1.1. Nucleation agent theory:Nucleation agent theory: Non-collagenous proteins of the matrix provide Non-collagenous proteins of the matrix provide

'nucleation' sites of the correct geometry for 'nucleation' sites of the correct geometry for deposition of calcium phosphate.deposition of calcium phosphate.Note: Note:

Calcium phosphate is relatively insoluble in water and Calcium phosphate is relatively insoluble in water and precipitation of this salt can occur spontaneously if the precipitation of this salt can occur spontaneously if the concentrations of calcium, phosphate, or both, cause the concentrations of calcium, phosphate, or both, cause the 'solubility product' of calcium phosphate to be exceeded 'solubility product' of calcium phosphate to be exceeded

The solubility product for calcium phosphate is:The solubility product for calcium phosphate is: KKsp = [Casp = [Ca2+2+]]33[PO[PO44

3-3-]]22

It is pH dependent, and at pH 7.0 its value is 25.It is pH dependent, and at pH 7.0 its value is 25.

2.2. ‘‘Matrix vesicles’ theory:Matrix vesicles’ theory:

• Osteoblasts and chondrocytes acquire calcium Osteoblasts and chondrocytes acquire calcium

and phosphate ions, form concentrated calcium and phosphate ions, form concentrated calcium

phosphate at the cell periphery and exfoliate it as phosphate at the cell periphery and exfoliate it as

vesicles (which also contain alkaline phosphate) vesicles (which also contain alkaline phosphate)

prior to its deposition on collagen, and other prior to its deposition on collagen, and other

proteoglycans secreted by osteoblastsproteoglycans secreted by osteoblasts

Both theories need enough calcium and Both theories need enough calcium and phosphatephosphate.. • Calcium and phosphate ion concentrations in Calcium and phosphate ion concentrations in

the aqueous medium may be altered by binding the aqueous medium may be altered by binding to proteins, to proteins,

• Complex localized concentration changes may Complex localized concentration changes may occur as a result of metabolic processes. occur as a result of metabolic processes.

• Furthermore, the 'micro-environmental' Furthermore, the 'micro-environmental' concentrations of calcium and phosphate in concentrations of calcium and phosphate in biological systems may not be identical to those biological systems may not be identical to those in the bulk of the solution.in the bulk of the solution.

The structural strengthThe structural strength (and therefore its (and therefore its mechanical function) of any bone is mechanical function) of any bone is determined by the following parameters:determined by the following parameters: The volume of bone matrixThe volume of bone matrix The type of bone matrix (woven or lamellar, )The type of bone matrix (woven or lamellar, ) The degree of mineralization of the matrixThe degree of mineralization of the matrix The structural arrangement of the matrixThe structural arrangement of the matrix

Alterations in any of these parameters will Alterations in any of these parameters will result in a potentially mechanically result in a potentially mechanically dysfunctional skeleton.dysfunctional skeleton.